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

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.

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Chromosome count improvement and digitalization of Neolamarckia cadamba

10.21203/rs.3.rs-90777/v1 ◽

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.

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Regeneration of whole plants from cultured root tips of Limnophila indica

Canadian Journal of Botany ◽

10.1139/b81-133 ◽

1981 ◽

Vol 59 (6) ◽

pp. 969-973 ◽

Cited By ~ 11

Author(s):

Sunanda Rao ◽

H. Y. Mohan Ram

Keyword(s):

Liquid Medium ◽

Root Tip ◽

Root Tips ◽

Cortical Cells ◽

Starch Grains ◽

Histological Studies ◽

Shoot Buds ◽

Whole Plants

Root tips (1 cm long) were excised from in vitro grown plants of Limnophila indica (L.) Druce and cultured in liquid medium with 2% sucrose. The explants floated horizontally on the surface of the medium; they became green, and their tips started to grow geotropically into the medium. Four to six shoot buds each were differentiated in 84.5% of the cultures in 4 weeks. Subsequently, shoot buds covered the surface of the bent part of the root and portions above and below it. No buds were formed at the cut end or near the growing root tip. Buds failed to differentiate in roots in which the apex was not growing. Histological studies of the roots showed that the buds originated from proliferating cortical cells, which had prominent chloroplasts and starch grains. The shoot buds grew into entire plants which flowered eventually.

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Polyprenyl phosphate sugars synthesized during slime-polysaccharide production by membranes of the root-cap cells of maize (Zea mays)

Biochemical Journal ◽

10.1042/bj1780661 ◽

1979 ◽

Vol 178 (3) ◽

pp. 661-671 ◽

Cited By ~ 19

Author(s):

J R Green ◽

D H Northcote

Keyword(s):

Endoplasmic Reticulum ◽

Zea Mays ◽

Neutral Lipids ◽

Root Tip ◽

Synthetase Activity ◽

Root Tips ◽

Maize Roots ◽

Glucose Derivatives

Two types of experiments were carried out; either maize roots were incubated in L-[1-3H]fucose or membranes were prepared from root tips and these were incubated with GDP-L-[U-14C]fucose or UDP-D-[U-4C]glucose. The radioactively labelled lipids that were synthesized in vivo and in vitro were extracted and separated into polar and neutral components. The polar lipids had the characteristics of polyprenyl phosphate and diphosphate fucose or glucose derivatives, and the neutral lipids of sterol glycosides (fucose or glucose). A partial separation of the glycolipid synthetase reactions was achieved. Membranes were fractionated into material that sedimented at 20,000g and 100,000g. Most of the polar glycolipid synthetase activity (for the incorporation of both fucose and glucose) was located in the 100,000 g pellet, and this activity was probably located in the endoplasmic reticulum. The neutral lipid, which contained fucose, was synthesized mainly by membranes of the 20,000g pellet, and the activity was probably associated with the dictyosomes, whereas the neutral glucolipids were synthesized by all the membrane fractions. It is suggested that the polar (polyprenyl) lipids labelled with fucose could act as possible intermediates during the synthesis of the glycoproteins and slime in the root tip.

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Improved Method of Enzyme Digestion for Root Tip Cytology

HortScience ◽

10.21273/hortsci12024-17 ◽

2017 ◽

Vol 52 (7) ◽

pp. 1029-1032 ◽

Cited By ~ 2

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.

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New chromosome numbers in Pilocarpus Vahl (Rutaceae)

Acta Botanica Brasilica ◽

10.1590/s0102-33062000000100002 ◽

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.

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

Canadian Journal of Genetics and Cytology ◽

10.1139/g83-077 ◽

1983 ◽

Vol 25 (5) ◽

pp. 513-517 ◽

Cited By ~ 15

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.

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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. 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.

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Heat stress tolerance assayed in four wine-producing grapevine varieties using a cytogenetic approach

Ciência e Técnica Vitivinícola ◽

10.1051/ctv/20193401061 ◽

2019 ◽

Vol 34 (1) ◽

pp. 61-70

Author(s):

Ana Carvalho ◽

Fernanda Leal ◽

Manuela Matos ◽

José Lima-Brito

Keyword(s):

Cell Cycle ◽

Heat Stress ◽

Mitotic Cell ◽

Root Tip ◽

Mitotic Cell Cycle ◽

Root Tips ◽

Excessive Heat ◽

Dividing Cells ◽

Treatment Interaction

The degree of tolerance to heat stress (HS) differs among grapevine varieties. HS affects the duration and the regularity of the cell cycle in plants. The cytogenetic studies in grapevine are scarce, and the consequences of HS in the mitosis are barely known. This work intends to evaluate the consequences of induced HS in the mitotic cell cycle and chromosomes of four wine-producing varieties: Touriga Franca (TF), Touriga Nacional (TN), Rabigato and Viosinho using a cytogenetic approach. HS (1h at 42 ºC) was induced in plants of the four grapevine varieties that grew in vitro for 11 months. Plants of the same varieties and with equal age were used as control (maintained in vitro at 25 ºC). Three plants per variety and treatment (control and HS) were analysed. After HS, root-tips were collected in all plants and immediately fixed to be used for the preparation of mitotic chromosome spreads. In total, 6,116 root-tip cells were scored. Among the 5,973 dividing cells, 24.33% showed different types of irregularities in all mitotic phases (prophase, metaphase, anaphase, and telophase). After HS, the mitotic index (MI) decreased in the varieties TF and Viosinho, and increased in TN and Rabigato, relatively to the control. However, the average values of MI did not show statistically significant differences (p ˃ 0.05) among varieties, treatments and for the variety x treatment interaction. The percentage of dividing cells with anomalies (%DCA) increased after HS in all varieties relatively to the control. The average values of %DCA presented statistically significant differences (p < 0.05) only between treatments. As far as we know, this work constitutes the first cytogenetic evaluation of the HS effects in the mitotic cell cycle and chromosomes of grapevine using meristematic cells of root-tips. TN has been considered tolerant to various abiotic stresses (drought and excessive heat and light) based on other methodologies. TF and Viosinho have been referred as tolerant to abiotic stress without deeper studies available, and till the development of our investigation, the sensibility of Rabigato was unknown. Among the varieties analysed in this work, Rabigato revealed to be the less tolerant to HS. This research can be useful for selection of grapevine clones more tolerant to HS for commercialization and for the improvement of the economic sustainability.

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Induksi Kalus serta Regenerasi Tunas dan Akar Cabai melalui Kultur In Vitro

Jurnal AgroBiogen ◽

10.21082/jbio.v6n2.2010.p65-74 ◽

2016 ◽

Vol 6 (2) ◽

pp. 65

Author(s):

Ifa Manzila ◽

Sri H Hidayat ◽

Ika Mariska ◽

Sriani Sujiprihati

Keyword(s):

Callus Induction ◽

Embryogenic Callus ◽

Adventitious Shoots ◽

Leaf Explants ◽

Root Tip ◽

Root Induction ◽

Ms Medium ◽

Root Tips ◽

Culture Bottle

Callus Induction and Regeneration of Shoot and Root of Chill through In Vitro Culture. Ifa Manzila, Sri H. Hidayat, Ika Mariska, and Sriani Sujiprihati. In vitro culture is one way for a fast and effective plant propagation. This method is also useful for preliminary selection of plant resistance to disease, including the chili. In vitro propagation method for chili has not been widely reported. A study was conducted to obtain effective techniques for callus induction and regeneration into shoots on three red chili cultivars (cv) Gelora, Sudra, and Chili 109. The study consisted of four activities, namely the induction of callus formation, induction of embryogenic callus, callus regeneration into adventitious shoots, and root induction from the adventitious shoots. Murashige Skoog (MS) medium + 0.6% agar + 3% sucrose were used as basal medium, 20 ml/bottle. Young leaves, hypocotyls and root tips of 21-day-old chili seedlings were used as sources of explants. Each experiment was arranged in a completely randomized design with 10 replications, one culture bottle for each treatment. The callus induction experiments using the explants of young leaf explants, hypocotyl, and root tips were done separately. Each treatment consisted of explants from the three chili cultivars on MS medium containing three composition of growth regulators (PGR) BAP + NAA, 10 explants/bottle. The embryogenic callus induction was conducted by growing the callus in bottles containing a medium that contains three compositions PGR 2,4-D + thidiazuron 0.5 mg/l. Induction of shoot formation was done by growing the embryogenic callus on medium containing three composition of plant growth regulator BAP + NAA. Induction of root formation was performed by growing adventitious shoots on ½ MS and 1 MS medium + NAA 0.5 to 1.0 mg/l. The results showed that young leaves are the best explant source for callus and shoot formations in chili through tissue culture compared with the hypocotyl and the tip. Gelora is the most responsive chili cultivar to callus, shoots, and roots formation of in their respective medium, compared to Sudra and Chile 109. MS medium containing BAP 3-7 mg/ml and NAA 1 mg/ml can be used to induce the growth of callus from young leaf explants, hypocotyl and seedling root tip chili cv Gelora, Sudra, and Chile 109, but its growth was very slow and did not produce embryogenic callus. Embryogenic callus formation can be induced by both non-embryogenic callus Hak Cipta © 2010, BB-Biogen growing the callus on MS medium containing 2,4-D 3 mg/l + thidiazuron 0.5 mg/ l. Formation of callus that can regenerate into shoots should use an MS medium containing 2,4-D 3 mg/l + thidiazuron 0.5 mg/ l followed by subculture on MS medium + BAP 3 mg/l + thidiazuron 0.5 mg/l to induce shoot elongation. Medium ½ MS and 1 MS containing NAA 0.5-1.0 mg/l can be used to induce root formations on shoot culture of chili cv Gelora but not for cv Chili 109.

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