Chromosome count improvement and digitalization of Neolamarckia cadamba

2020 ◽  
Author(s):  
Wee-Hiang Eng ◽  
Wei-Seng Ho ◽  
Kwong-Hung Ling
Keyword(s):  
Open Source ◽  
Digital Technology ◽  
Chromosome Count ◽  
Root Tip ◽  
Root Tips ◽  
Chromosome Counts ◽  
Laboratory Equipment ◽  
The Public ◽  
Chromosome Staining ◽  
Graphic Software

Abstract Background: Chromosome count is the only direct way to determine the number of chromosome of a species. This study is often considered trivial that seldom described and discussed in detail. Therefore, it is evitable that chromosome count protocol should be revised and revisited before it becomes obliterated. Our chromosome counts in N. cadamba root tips have encountered challenges that prevent us from obtaining clear micrograph with the correct chromosome number. Several obstacles were determined through micrograph observation such as existing unwanted particles in cells, poor chromosome staining and chromosome crumping. Results: The chromosome counts of Neolamarckia cadamba under optimized procedure yielded 44 chromosomes per nucleus. To overcome these, root tip types, stain types, squashing methods were among factors assessed to obtain clear micrograph. We also apply digital technology in term of online database and graphic software that are open source and freely accessible to the public. Conclusions: Throughout the study, we used a few basic laboratory equipment and chemicals, thus making this study economical and applicable in a basic laboratory. The availability of online digital software and databases provide open-source platforms that will ease the efforts in chromosome count.

scholarly journals Cytogenetic, chromosome count optimization and automation of Neolamarckia cadamba (Rubiaceae) root tips derived from in vitro mutagenesis

2021 ◽  
Vol 13 (3) ◽  
pp. 10995
Author(s):  
Wei-Seng HO ◽  
Wee-Hiang ENG ◽  
Kwong-Hung LING
Keyword(s):  
Open Source ◽  
Chromosome Count ◽  
Root Tip ◽  
In Vitro Mutagenesis ◽  
Root Tips ◽  
Chromosome Counts ◽  
Laboratory Equipment ◽  
Online Databases ◽  
Chromosome Staining

Chromosome count is the only direct way to determine the number of chromosomes of a species. This study is often considered trivial that seldom described and discussed in detail. Therefore, it is inevitable that the chromosome count protocol should be revised and revisited before it becomes obliterated. In the present study, we encountered challenges in obtaining a clear micrograph for the chromosome count of active mitotic cells of Neolamarckia cadamba (Roxb.) Bosser (Rubiaceae) root tips. Several obstacles were determined through micrograph observation, such as existing unwanted particles in cells, poor chromosome staining and chromosome clumping. To overcome these, root tip types, staining methodologies, squashing methods were among the factors assessed to obtain clear micrographs. The chromosome counts of N. cadamba under optimized procedure showed 2n = 44 chromosomes. We also apply digital technology in chromosome counts, such as online databases and graphic software that are open source and freely accessible to the public. Only basic laboratory equipment and chemicals were used throughout the study, thus making this study economical and applicable in a basic laboratory. The availability of online digital software and databases provide open-source platforms that will ease the efforts in chromosome count.

scholarly journals Improved Method of Enzyme Digestion for Root Tip Cytology

HortScience ◽  
2017 ◽  
Vol 52 (7) ◽  
pp. 1029-1032 ◽  
Author(s):  
Jason D. Lattier ◽  
Hsuan Chen ◽  
Ryan N. Contreras
Keyword(s):  
Chromosome Numbers ◽  
Enzyme Digestion ◽  
Root Tip ◽  
Root Tips ◽  
Chromosome Counts ◽  
Ploidy Levels ◽  
Long Duration ◽  
Wide Range ◽  
Plant Sciences ◽  
Breeding And Genetics

Chromosome numbers are an important botanical character for multiple fields of plant sciences, from plant breeding and genetics to systematics and taxonomy. Accurate chromosome counts in root tips of woody plants are often limited by their small, friable roots with numerous, small chromosomes. Current hydrolysis and enzyme digestion techniques require handling of roots before the root squash. However, optimum chromosome spread occurs when the cell walls have degraded past the point of easy handling. Here, we present a new enzyme digestion protocol that is fast, efficient, and flexible. This protocol reduces handling of the roots allowing for long-duration enzyme digestion. Digestions are performed on a microscope slide, eliminating the need for handling digested cells with forceps or pipettes. To illustrate the flexibility of this method across woody plant taxa, we performed chromosome counts on five angiosperms and one gymnosperm. Ploidy levels included diploids, triploids, and tetraploids with chromosome numbers ranging from 2n = 16 to 2n = 80. The range of holoploid 2C genome sizes spanned 1.54–24.71 pg. This protocol will provide a useful technique for plant cytologists working with taxa that exhibit a wide range of genome size and ploidy levels.

scholarly journals New chromosome numbers in Pilocarpus Vahl (Rutaceae)

2000 ◽  
Vol 14 (1) ◽  
pp. 11-24
Author(s):  
Ladislau A. Skorupa
Keyword(s):  
Chromosome Numbers ◽  
Room Temperature ◽  
Basic Number ◽  
Root Tip ◽  
Root Tips ◽  
Chromosome Counts ◽  
Pre Treatment ◽  
First Time ◽  
Tip Cells ◽  
Root Tip Cells

Chromosome counts for eight species of Pilocarpus Vahl (Rutaceae) a native of Brazil are reported for the first time. Chromosome numbers were determined from mitotic root tip cells of seedlings derived from field collections and grown in the greenhouse. Feulgen staining was used. Initial pre-treatment of root tips was done by using a saturated aqueous solution of alpha-bromonapthalene for two hours at room temperature (20-25ºC). Chromosome numbers of 2n=44 and 2n=88 were determined for the examined taxa. The present results suggest the occurrence of tetraploidy in P. spicatus St.-Hil. and P. carajaensis Skorupa, and a possible basic number x=22 to the genus Pilocarpus.

Cytogenetic characteristics of wheat plants regenerated from anther calli of 'Centurk'

1983 ◽  
Vol 25 (5) ◽  
pp. 513-517 ◽  
Author(s):  
Dalia T. Kudirka ◽  
Gideon W. Schaeffer ◽  
P. Stephen Baenziger
Keyword(s):  
Chromosome Doubling ◽  
Doubled Haploids ◽  
Triticum Aestivum L ◽  
Root Tip ◽  
Anther Wall ◽  
Root Tips ◽  
Chromosome Counts ◽  
Spontaneous Chromosome ◽  
Tip Cells ◽  
Root Tip Cells

Plants were recovered from 10 different calli derived through anther culture of Triticum aestivum L. em Thell (cv. 'Centurk'). Chromosome counts and estimates of ploidy level were made on cells from roots of these developing plants. Plants with polyhaploid cells were regenerated from all calli indicating that they were of microspore origin. Three populations of plants were recognized: first, those that were polyhaploid and euploid; second, those that were almost totally polyhaploid but aneuploid; and third, those plants which were largely hexaploid with some cells reflecting spontaneous chromosome doubling. Aneuploid cells with corresponding polyhaploid and hexaploid chromosome numbers in root-tip cells among plants regenerated from a number of calli were taken to indicate that these cells were doubled haploids and not hexaploid cells of the anther wall. Mixoploid plants were regenerated from secondary calli in which chromosome doubling was known to have been induced at the callus stage. The presence of individual mixoploid root tips in these plants was assumed to indicate that individual organs of a plant may arise from several cells of a callus.

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

2016 ◽  
Vol 21 (2) ◽  
pp. 113 ◽  
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 />

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

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.

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

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.

scholarly journals Molecular Identification of Ectomycorrhizal Mycelium in Soil Horizons

2003 ◽  
Vol 69 (1) ◽  
pp. 327-333 ◽  
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.

Proteomic Analyses of Soybean Root Tips During Germination

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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