Flow Cytometry Applied in Tissue Culture

Pasqual, Moacir; Pio, Leila Aparecida Salles; Oliveira, Ana Catarina Lima de; Soares, Joyce Dória Rodrigues

doi:10.5772/50986

Cited by 7 publications

(7 citation statements)

References 41 publications

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“…The nuclei were then stained by adding 25 μl of 1 mg/ml propidium iodide to each sample. To compare DNA content in picograms, two other species, Pisum sativum with 9.09 pg (Pasqual et al, 2012) and Solanum lycopersicum with 1.86 pg were used as external reference standards, using the same procedure for nucleus suspension. For each sample, 10,000 nuclei were evaluated through a logarithmic scale.…”

Section: Dna Content Estimate By Flow Cytometrymentioning

confidence: 99%

“…Flow cytometry is used to characterize vegetal material for several purposes, such as DNA content analysis, ploidy verification, and cell cycle acquisition (Ochatt, 2008). Specifically, in tissue culture, this technique has been important to verify genetic stability, identify hybrids, check ploidy, and quantify genome size (Doležel and Greilhuber, 2010b;Pasqual et al, 2012). Growth regulators through in vitro culture, may cause somaclonal variations which might be from genetic or epigenetic (Miguem and Marum, 2011;Georgiev et al, 2014); this mechanism regulation influences the genetic expression affecting the phenotype.…”

Section: Introductionmentioning

confidence: 99%

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Growth regulators, DNA content and anatomy in vitro-cultivated Curcuma longa seedlings

Antoniazzi¹,

Ferrari²,

Nascimento³

et al. 2016

Afr. J. Biotechnol.

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Curcuma longa L., from the Zingiberaceae family, generally reproduces through its rhizomes, which are also utilized for therapeutic purposes because they are rich in terpenoids. Its conventional propagation has low efficiency due to the small number of seedlings and their contamination by pathogens. Therefore, this study aimed to evaluate the influence of growth regulators on the development of in vitro-cultivated C. longa as well as to determine their influence on DNA content and foliar anatomy. Shoots were inoculated in MS culture medium with the addition of 30 g/L of sucrose and 6.5 g/L of agar, and a pH adjusted to 5.8. Two assays were built to study the multiplication and rooting phases of growth. The first assay evaluated the influence of eight concentrations of cytokinins and auxins on the multiplication phase. Leaf samples were analyzed for DNA content through flow cytometry, utilizing two reference standards, green pea, and tomato. Characteristics of leaf anatomy were also measured in four time periods. The second assay analyzed the influence of six auxin concentrations on the rooting phase. The first assay showed that the root systems grew more in treatment 3 (4.44 µM benzylaminopurine [BAP], 0.46 µM kinetin [KIN]) and reached greater dry mass in T8 (8.88 µM BAP, 0.92 µM KIN, 2.16 µM naphthalene acetic acid [NAA]). The largest fresh matter of the main shoot was found in T2 (4.44 µM BAP). The estimated DNA content varied depending on the presence of supplemental growth regulators, from 2.38 to 2.77 pg, and was greater in T4 (4.44 µM BAP, 1.08 µM NAA) and T5 (4.44 µM BAP, 0.46 µM KIN, 1.08 µM NAA). Results from the latter two treatments were not significantly different. Estimates of DNA content were precise, as indicated by coefficients of variation that were much lower than 5%. The results also showed quantitative variation of evaluated anatomical traits. In general, there was a thin epidermis layer with rectangular cells, followed by parenchyma with octahedral cells and differentiated xylem and phloem. In leaf parenchyma, the presence of idioblasts containing phenolic compounds was observed in all growth stages. In the rooting phase, the supplementary auxins affected the dry matter of the aerial part and roots. The highest averages were observed in treatments with 2.0 µM NAA.

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Section: Dna Content Estimate By Flow Cytometrymentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Growth regulators, DNA content and anatomy in vitro-cultivated Curcuma longa seedlings

Antoniazzi¹,

Ferrari²,

Nascimento³

et al. 2016

Afr. J. Biotechnol.

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“…Chromosome variability of cells in vitro is among the best studied cytological disorders, like endomitosis and endoreduplication whicht causes polytene chromosomes formation, which are formed due to repeated DNA synthesis without further nucleus and cells fission (Carvalheira, 2000). Therefore, cytometric monitoring is carried out to choose appropriate medium (Pasqual et al, 2012). Reduced number of unwanted mutations and non-hereditary changes can be achieved by adjusting the concentration of exogenous phytohormones introduced in the nutrient medium instead of those regulating normal cell division in vivo.…”

Section: Variant 1 Control (No Dye)mentioning

confidence: 99%

“…That is why cytometric control is mostly limited to establishing ploidy level and chromosomal aberrations. Cells with unbalanced number of chromosomes in the callusogenesis may arise in connection with indirect division in the callusogenesis (Dhooghe et al, 2011;Pasqual et al, 2012). Scientists of the Institute of Bioenergy Crops and Sugar Beet of National Academy of Agrarian Sciences have developed a method that allows the analysis to be performed of genome ploidy level variability of initial breeding materials of sugar beet with "Partec" ploidyanalyzer technology.…”

Section: Mediummentioning

confidence: 99%

In vitro rhizogenesis of sugar beet microclones

Полищук

Каrpuk

Mykolaiko

et al. 2017

Regul. Mech. Biosyst.

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The features of rhizogenesis of male-sterile simple hybrids, maternal components of sugar beet heterozosis hybrid and O-types of Darinka variety grown from seeds were investigated. The paper presents the results of studying the main basic nutrient media (B5–A1, B5–A2, B5–A3, B5–A4, B5–A5 and B5–A6) for rhizogenesis, in which the content of macro- and microelements has been reduced by three times, the content of sucrose is reduced by almost 6 times, the amount of agar is reduced by almost five times, while the vitamin РР content is increased by 1.6 times, and the BAP is completely removed. The largest number of rooted microclones was obtained with NAAs by the high indexes of average number of roots and medium indexes of average roots length. To the second, by the number of rooted microclones is assigned, media of 2,4–D, 2,4–DB containing, potassium salt of NAA and IBA were classified, while IAA was characterized by a lower efficiency. The possibilities of regulation of growth and development processes of sugar beet in vitro explants in favor of undifferentiated growth at the stage of proliferation or organogenesis (hemo- and rhizogenesis) have been determined. To improve the quality of rooting of sugar beet plants-regenerants, we have developed the method for dimming agarized nutrient medium with methylene blue at a 0.05% concentration, which made it possible to reduce the inhibitory effect of light on the development of lateral roots. The average index of micro-roots rooting for MS-forms was 81.5 %. The technology of adaptation of micro-roots rooting was developed, in which the plant material at the initial stage of growth had slight morphological changes in the puffer apparatus and stem, but by the end of the vegetation, the plants acquired the appearance characteristic of the donor plants of explants. The clone’s resiliency is reached about 100% in the soil. According to the variability of morphological characteristics in reproduced in vitro sugar beet plants of the MS-forms of the Darynka hybrid, all the plants grown from the regenerants were more, than seed progeny from seed sowing of the same component obtained as a result of the attachment of sterility to the corresponding O-type. It has been found that in vitro cultivation has not weakened, and in some cases even increased the mitotic index of meristem cells of young roots of sugar beet. Most genotypes have reduced the number of pathological mitoses, apparently due to the activation of reparative systems, but did not extend the duration of individual phases of mitosis; without increasing the number of anaphase with bridges and fragments, but changed the number of anaphases with other chromosomal abnormalities. In all variants of the experiment, where activation of growth processes has been observed, the lowest variability of the signs is observed, that is, under the influence of stimulants, which are part of nutrient media, obviously there is unification of cell populations.

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“…Genetic uniformity of the plants can be detected using many techniques, such as cytogenetic analysis, flow cytometry and/or molecular markers such as amplified fragment length polymorphism (AFLP), inter simple sequence repeats (ISSR), isozymes, random amplified polymorphic DNA (RAPD), restriction fragment length polymorphisms (RFLP) and simple sequence repeats (SSR) [13,14]. Among these techniques, flow cytometry has been shown to be an easy, rapid, accurate and economical method for screening the genetic stability of propagated plants [15][16][17][18]. Flow cytometry can be complemented by traditional cytological studies, including chromosome counting.…”

Section: Introductionmentioning

confidence: 99%

In vitro propagation of Silene bolanthoides Quézel, Contandr. & Pamukç. and assessment of genetic stability by flow cytometry

Çördük¹,

Yücel²,

Akıncı³

et al. 2018

ARCH BIOL SCI BELGRA

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Silene bolanthoides Quézel, Contandr. & Pamukç. is an endemic species from Kazdagi (Mt. Ida), Canakkale-Balikesir, Turkey. In order to develop an efficient shoot regeneration protocol, the leaf, nodal and internodal explants of S. bolanthoides were cultured on Murashige and Skoog (MS) medium containing benzyladenine (BA) alone or in combination with α-naphthaleneacetic acid (NAA). The highest number of regenerated shoots (5.75±0.1) was obtained from nodal explants that were cultured on MS medium with 8.8 µM BA+0.54 µM NAA. Regenerated shoots were rooted on MS medium without plant growth regulators (PGRs). Rooted plants (2-3 cm) were separately transferred to pots containing a mixture of peat and perlite (3:1 v/v) and acclimatized successfully in a growth chamber. Genetic stability of the propagated plants was assessed by flow cytometry and cytological analysis. Flow cytometry analysis demonstrated that regenerated plants had 2.61±0.01 pg nuclear DNA (2C) and seed-derived plants had on average 2.58±0.02 pg/2C. Cytological analysis showed that the regenerated plants had the same chromosome number as seed-derived plants of S. bolanthoides (2n=24). It was determined that regenerated plants were uniform in chromosome number and had a similar DNA content to the seed-derived ones, indicating that the described efficient shoot regeneration protocol can be applied for ex situ conservation of this species.

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Flow Cytometry Applied in Tissue Culture

Cited by 7 publications

References 41 publications

Growth regulators, DNA content and anatomy in vitro-cultivated Curcuma longa seedlings

Growth regulators, DNA content and anatomy in vitro-cultivated Curcuma longa seedlings

In vitro rhizogenesis of sugar beet microclones

In vitro propagation of Silene bolanthoides Quézel, Contandr. & Pamukç. and assessment of genetic stability by flow cytometry

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