Accumulation of cadmium and zinc in barley regenerants on a provocative soil background with cadmium

Background. An effective way to obtain barley (Hordeum vulgare L.) genotypes stress-tolerant to cadmium, with a low level of toxic ion accumulation in grain, is the selection of cells in selective in vitro systems, based on somaclonal variability, which promotes the formation of specific and nonspecific adaptive mechanisms.Materials and methods. The object of the study was cv. ‘999-93’, developed by selection from a hybrid combination of spring barley (Luly × Conrad) × 2867-80, and its regenerated forms in seed reproductions of the 3–5th generation, induced in the process of cell selection on media with cadmium, aluminum and polyethylene glycol. The plants were grown under normal soil conditions and against a provocative background for cadmium.Results. The contribution of the environment-forming activity in the roots of the studied genotypes to inactivation of toxic ions appeared insignificant. The total removal of cadmium by plants against a provocative background increased 22.5 times, reaching 5.8–10.3 mg/kg of dry phytomass when distributed among organs: roots (91.9–93.4%) > stems (5.9–7.8%) > grain (0.5–0.8%). The amount of toxic metal in grain increased 11 times in the original form and 2.8–6.8 times in regenerants. The negative effect of excess cadmium in the soil on the accumulation of zinc in barley was shown. There was no gradation in the importance of organs for zinc accumulation or any presence of functional barriers preventing this. Regenerants induced on selective media with cadmium had the greatest adaptive advantages to stress: pronounced barrier functions of roots, minimal accumulation of toxic ions in aerial organs, and high seed productivity (they exceeded the original genotype by 35.5%). Adaptive reactions associated with the limitation of cadmium accumulation in plant tissues of regenerants, obtained by in vitro selections with aluminum and an osmotic, were shown to be weak.

Genetic identity of mint cultivars after in vitro conservation, assessed by ISSR primers

The species of the genus Mentha have been known since ancient times and have significant value in the pharmaceutical, cosmetic and food industries, as well as in medicine. For the widespread use of mint, including in a variety of breeding programs, and the preservation of genetic diversity, effective methods of maintaining cultivars and collection samples are required. Thanks to the development of biotechnological methods, in particular, the creation of slow-growing collections, are now actively used as an effective alternative to field collections. It is known that the cultivation of tissues and organs on artificial culture media can cause somaclonal variability. The purpose of this work is to study the effect of in vitro storage at 4-6°C without illumination after 3 and 4 in vitro conservation cycles on the genetic stability of three cultivars of mint Azhurnaya, Bergamotnaya and Zagrava using ISSR primers. 1 cycle: 1 year of in vitro conservation, microcutting and 2 subcultures of regrowth in a culture room. After conservation, the number of viable explants was 70.0-82.1%. Callus formation at the base of the shoots was not observed in any of the cultivars. After 3 and 4 cycles of in vitro conservation, genetic stability was assessed using 11 ISSR primers. It was found that all three mint genotypes showed full compliance (length and number of amplicons) with the profiles of control samples for all studied markers. It was also found that the markers used by ISSR are highly informative for mint cultivars.

Regeneration from leaf explants of steppe cherry (Prunus fruticosa Pall)

The article represents data on morphogenesis from leaf explants of three steppe cherry genotypes, as well as the degree of somaclonal variability at in vitro and ex vitro stages, and in the field. It was revealed that a content of 6-benzylaminopurine, 4.43 μM, in combination with auxin, 0.5–0.6 μM, stimulates in the light the direct organogenesis in the tissues of the leaf base. This reaction was observed from 16.7 to 75.0% of explants, depending on the genotype. An equal 6-benzylaminopurine - auxin ratio (1: 1) led to the callus along with microshoots. Depending on the genotype, up to 30.0% of explants had such a mixed type of organogenesis. The mitotic index value in the apical leaflets differed depending on the day time. At the stage of micropropagation itself, an increase of the mitotic index was observed from 10 to 16 hours; at the stage ex vitro, no significant differences in the mitosis frequency were revealed within this time interval. No significant differences were found between the level of the mitotic index for plants obtained directly from leaves and those from buds. The mitosis passed without disturbances. No phenotypic changes in plant habit, shape and color of leaves were found.

Creation of clary sage cultivar using cell engineering methods. 1. Obtaining of plant-regenerants in callus culture in vitro

To increase the efficiency in agricultural plant breeding, including clary sage – one of the main essential oil crops grown in Russia, it is necessary to use biotechnological methods. One of these techniques is based on the induction of somaclonal variability in the callus tissue culture. To develop it, it is necessary to optimize the conditions for obtaining plant-regenerants in vitro and their analysis. The aim of this work was to study the features of morphogenesis and regeneration of plants from callus cultures to develop cell technologies for creating an initial breeding material based on somaclonal variability in Salvia sclarea L. In the course of the research, we found that the optimal explants for obtaining morphogenic callus, from which shoots were regenerated, were segments of buds and stems with a node (isolated from seedlings in vitro). Cytological analysis of callus cultures revealed two types of morphogenesis – organogenesis (gemmogenesis) and somatic embryogenesis. The features of the morphogenic callus formation of six sage cultivars and samples during the long-term cultivation were studied. The maximum frequency of morphogenesis was noted in the 2nd passage (from 32.4 to 85.2 %, depending on the genotype). Then, to the 8–10th passage, this indicator decreased to 0.0–3.9 %.‘S-785’ and ‘Taigan’ cultivars showed the highest morphogenesis frequency (81.5–85.2 %) and duration of callus regeneration potential (up to the 10th passage). The analysis of callus cultures of six donor plants of ‘S-785’ cultivar helped us to reveal their heterogeneity in morphogenesis induction ability. The maximum frequency of morphogenic callus formation (76.3–91.5 % in the 2nd passage) and the duration of the morphogenic potential preservation (up to the 12th passage) were observed in plants No. 3 and 9, whereas in No. 2, regeneration with a frequency of 3.6–9.7 % was observed only during three passages. Analysis of plants obtained from calli showed their variability in morphology – up to 12.5 % of the samples had deviations compared to the initial cultivar ‘S-785’ in leaf shape, inflorescence structure, flower color, etc. Somaclonal changes in morphological and economically useful traits revealed in regenerants indicate that they are promising for use in sage breeding.

The prospect of using clonal micropropagation to conserve the gene pool of natural Tulipa suaveolens (Liliaceae) populations

Despite the morphological correspondence between the regenerated and natural Tulipa suaveolens plants, the ISSR analysis identified a relatively high degree of somaclonal variability between them.

Somaclonal variability of conifers in culture in vitro

The use of somatic embryogenesis is one of the promising methods of conifer propagation on an industrial scale. However, this technology has a number of problems, which include the appearance of somaclonal variation in cell and tissue culture. The review considers the causes and methods for detecting somaclonal variability of conifer in culture in vitro. It is shown that it is necessary to use a complex of molecular, cytogenetic, morphological, physiological methods for the analysis of somaclonal changes in embryogenic plant cultures.

Possible replacement of cytokinins and auxins in culture in vitro

Aim. To perform an analysis of the effectiveness of our proposed modification of the classical MS medium in which auxin and cytokinin are replaced by derivatives of the class of tetrahydrothio-fendioxide and pyridine. Methods. In this study, the work with cell culture in vitro, in particular aseptic sprouting of seeds, microclonal reproduction, callusogenesis and initiation of various types of morphogenesis was carried out according to known techniques. Two varieties of potatoes (Slovyanka, Lugovskaya) and two varieties of tomato (Lagidny, Bobrytsky) were used to induce somaclonal variability. Results. The ability of preparations of tetrahydrothiophenedioxide and pyridine derivatives to perform the function of auxins and cytokinins in the MS medium in the callusogenesis of Solanacea cultures in vitro was established. Conclusions. It has been shown that with the use of the new generation drug (tetrahydrothiophenedioxide-pyridine), as a substitute for auxin and cytokinin in the MS medium, the growth of callus tissue is even increased. The production of the pilot batch of this substitute proved to be much cheaper than synthesis or the purchase of phytohormones. Keywords: modification of MS medium, phytohormones substitute, tetrahydro-thiophenedioxide-pyridine, potato, tomato.

Effect of Different DNA Demethylating Agents on In vitro Cultures of Peach Rootstock GF 677

The appearance of somaclonal variability induced by in vitro cultivation is relatively frequent and, in some cases, provides a valuable source of new phenotypes suitable for crop improvement. Numerous studies have confirmed that these changes can be explained by alterations of DNA methylation. Interestingly, a group of chemical compounds termed ‘demethylating agents’ (DMT agents) enable artificial changes to be made in the DNA methylation state. Thus, these agents are theoretically able to induce new phenotypes or more favourable properties. The objective of the present study was to verify suitable conditions for the application of different DMT agents within in vitro protocols for micropropagation using the stone fruit rootstock GF 677 as an example. The impact of these agents on the properties of plant regenerants was evaluated, and their DNA methylation state was controlled by using an AFLP protocol based on a restriction endonuclease that differed in its sensitivity to methylated cytosines. Moreover, the effect of newly synthesised derivates was compared with that of conventional compounds with a well-documented DNA-demethylating impact. Based on the results, the suitable concentration for treatment by a DMT agent was established as approximately 50 µM. Promising results were generated using a combination of DMT agents with different mechanisms of action, such as azacytidine and dihydroxypropyladenine; under these conditions, probable synergy between methyltransferase interception by the cytosine analogue and interruption of methyl group donation by dihydroxypropyladenine significantly changed the DNA methylation state of treated plants. Regarding newly synthesised compounds, the 5,6-dihydro-5-azacytosine nucleoside showed the most promising results, which can likely be explained by its higher stability in the media used for in vitro cultivation. ********* In press - Online First. Article has been peer reviewed, accepted for publication and published online without pagination. It will receive pagination when the issue will be ready for publishing as a complete number (Volume 47, Issue 3, 2019). The article is searchable and citable by Digital Object Identifier (DOI). DOI link will become active after the article will be included in the complete issue. *********

The ploidy level of triticale plant regenerants, obtained by in vitro selection for resistance to abiotic stresses

Aim. To analyze the ploidy level of plant regenerants of winter triticale, obtained by in vitro selection for resistance to osmotic and salt stresses. Methods. By cytological analysis and flow cytometry methods there was determined the ploidy level in the plant regenerants of winter triticale obtained by in vitro selection for resistance to abiotic stresses. Results. The somaclonal variability of plant regenerants of winter triticale resistant to osmotic and salt stresses by ploidy level was observed. The cytological instability of resistant’s regenerants was revealed that was due in appearance of aneuploidy plants. Plants with aneuploid chromosome set (38–41) were characterized by reduced viability and abnormal generative organs resulting they are not formed normal ears and not received seeds. Conclusions. Among the obtained regenerants euploids were in most cases indicating a selective advantage of hexaploid cells in ability to morphogenesis. Keywords: Triticale, plant regenerants, cytological analysis, aneuploids, abiotic stresses.