scholarly journals Time of In Vitro Anther Culture May Moderate Action of Copper and Silver Ions that Affect the Relationship between DNA Methylation Change and the Yield of Barley Green Regenerants

Plants ◽  
2020 ◽  
Vol 9 (9) ◽  
pp. 1064
Author(s):  
Piotr T. Bednarek ◽  
Renata Orłowska
Keyword(s):  
Anther Culture ◽  
De Novo ◽  
Silver Ions ◽  
Dna Demethylation ◽  
Induction Medium ◽  
Green Plants ◽  
Ion Concentrations ◽  
Regenerated Plants ◽  
Short Time

Plant anther culture allows for the regeneration of uniform and homozygous double haploids. However, off-type regenerants may appear as a result of so-called tissue culture-induced variation (TCIV). In addition, the presence of Cu2+ and Ag+ ions in the culture medium might influence the number of green plants. The regenerants were obtained via anther cultures of barley under varying Cu2+ and Ag+ ion concentrations in the induction medium during distinct time conditions. DArTseqMet markers were evaluated based on regenerants and donor plants and delivering data on DNA demethylation (DM) and de novo methylation (DNM) and changes in methylation (Delta). The number of green regenerated plants per 100 anthers (GPs) was evaluated. The Cu2+ and Ag+ ion concentrations moderated relationships between Delta and the number of green plants conditional on time of tissue cultures. Depending on the ions, moderated moderation is valid within the different time of anther culture. When the highest concentration of copper is analyzed, plant regeneration is possible under short ‘Time’ (21 days) of anther culture wherein Delta is negative or under elongated Time when Delta is positive. Under 21 days of culture, the highest concentration of silver ions and when Delta is negative, some regenerants could be evaluated. However, under high Ag+ concentration when Time of culture is long and Delta positive, the highest number of green plants could be obtained.

scholarly journals Triticale Green Plant Regeneration Is Due to DNA Methylation and Sequence Changes Affecting Distinct Sequence Contexts in the Presence of Copper Ions in Induction Medium

Cells ◽  
2021 ◽  
Vol 11 (1) ◽  
pp. 84
Author(s):  
Renata Orłowska ◽  
Katarzyna Anna Pachota ◽  
Piotr Androsiuk ◽  
Piotr Tomasz Bednarek
Keyword(s):  
Plant Regeneration ◽  
Anther Culture ◽  
Sequence Variation ◽  
De Novo ◽  
Copper Ions ◽  
Green Plant ◽  
Dna Demethylation ◽  
Induction Medium ◽  
Regeneration Efficiency ◽  
De Novo Methylation

Metal ions in the induction medium are essential ingredients allowing green plant regeneration. For instance, Cu(II) and Ag(I) ions may affect the mitochondrial electron transport chain, influencing the Yang cycle and synthesis of S-adenosyl-L-methionine, the prominent donor of the methylation group for all cellular compounds, including cytosines. If the ion concentrations are not balanced, they can interfere with the proper flow of electrons in the respiratory chain and ATP production. Under oxidative stress, methylated cytosines might be subjected to mutations impacting green plant regeneration efficiency. Varying Cu(II) and Ag(I) concentrations in the induction medium and time of anther culture, nine trials of anther culture-derived regenerants of triticale were derived. The methylation-sensitive AFLP approach quantitative characteristics of tissue culture-induced variation, including sequence variation, DNA demethylation, and DNA de novo methylation for all symmetric-CG, CHG, and asymmetric-CHH sequence contexts, were evaluated for all trials. In addition, the implementation of mediation analysis allowed evaluating relationships between factors influencing green plant regeneration efficiency. It was demonstrated that Cu(II) ions mediated relationships between: (1) de novo methylation in the CHH context and sequence variation in the CHH, (2) sequence variation in CHH and green plant regeneration efficiency, (3) de novo methylation in CHH sequences and green plant regeneration, (4) between sequence variation in the CHG context, and green plant regeneration efficiency. Cu(II) ions were not a mediator between de novo methylation in the CG context and green plant regeneration. The latter relationship was mediated by sequence variation in the CG context. On the other hand, we failed to identify any mediating action of Ag(I) ions or the moderating role of time. Furthermore, demethylation in any sequence context seems not to participate in any relationships leading to green plant regeneration, sequence variation, and the involvement of Cu(II) or Ag(I) as mediators.

scholarly journals In Vitro Propagation of an Endangered Helianthus verticillatus by Axillary Bud Proliferation

Plants ◽  
2020 ◽  
Vol 9 (6) ◽  
pp. 712
Author(s):  
Marzena Nowakowska ◽  
Žaklina Pavlović ◽  
Marcin Nowicki ◽  
Sarah L. Boggess ◽  
Robert N. Trigiano
Keyword(s):  
Endangered Species ◽  
Shoot Proliferation ◽  
Genetic Fidelity ◽  
Induction Medium ◽  
Axillary Shoot ◽  
Axillary Shoot Proliferation ◽  
Regenerated Plants ◽  
Micropropagated Plants ◽  
Ssrs Markers

Helianthus verticillatus (Asteraceae), whorled sunflower, is a perennial species restricted to a few locations in the Southeastern United States. Habitat loss has caused H. verticillatus to become rare, and since 2014, it has been federally listed as an endangered species. As a part of the recovery plan for the restoration and protection of H. verticillatus, an efficient micropropagation protocol based on axillary shoot proliferation was developed. Various concentrations of 6-benzylaminopurine (BAP; 0 to 4.44 µM) were examined for their morphogenetic potential in the regeneration of six genotypes of H. verticillatus from the nodal explants derived from greenhouse-grown plants. Both the BAP concentration and genotype had significant effects on the regeneration capacity of H. verticillatus. Although the induced buds were observed on ½-strength Murashige and Skoog medium without plant growth regulators, a higher rate of induction and bud development were achieved on media with either 0.88 or 2.22 µM BAP, regardless of the genotype. Successful rooting of the induced shoots was achieved within four weeks after the transfer from the induction medium to the fresh ½-strength MS medium, but the rooting efficiency was dependent on the plant’s genetic background. Regenerated plantlets, with well-developed shoots and roots, were acclimatized successfully to greenhouse conditions with a 97% survival rate. Simple sequence repeats (SSRs) markers were employed to assess the genetic uniformity of the micropropagated plants of H. verticillatus. No extraneous bands were detected between regenerants and their respective donor plants, confirming the genetic fidelity and stability of regenerated plants. To our knowledge, the protocol developed in this study is the first such report for this endangered species.

scholarly journals Efficiency of anther culture technique in the production of wheat double haploids

2008 ◽  
pp. 35-40 ◽  
Author(s):  
Ankica Kondic-Spika ◽  
Borislav Kobiljski ◽  
Nikola Hristov
Keyword(s):  
Triticum Aestivum ◽  
Plant Regeneration ◽  
Anther Culture ◽  
Triticum Aestivum L ◽  
Green Plant ◽  
Culture Technique ◽  
Average Yield ◽  
Green Plants ◽  
Double Haploids

The objective of the study was to investigate efficiency of anther culture in the production of spontaneous double haploids from randomly selected heterozygous genotypes of wheat (Triticum aestivum L.). Anthers of 20 F1 wheat combinations were grown in vitro on a modified Potato-2 medium. All of the examined genotypes have shown the ability to produce pollen calluses as well as to regenerate green plants. On average for the whole experiment material, 47.2 calluses were produced per 100 cultured anthers. The green plant regeneration ranged from 0.8 to 13.4 green plants per spike, with an overall mean of 5.8. From the total of 582 regenerated green plants, 47.9% (279) were spontaneous double haploids. The final average yield from the study was 2.8 double haploids per spike.

The action of the Cu2+, Ag+ and time inducing the in vitro anther culture-derived barley regenerants

2020 ◽  
Author(s):  
Piotr Tomasz Bednarek ◽  
Renata Orłowska
Keyword(s):  
Dna Methylation ◽  
Tissue Culture ◽  
De Novo ◽  
Culture Conditions ◽  
Ion Concentration ◽  
Tissue Cultures ◽  
Regeneration Medium ◽  
Green Plants ◽  
Anther Cultures

Abstract BackgroundPlant regeneration via anther cultures is a world-wide approach as it allows for the regeneration of uniform and homozygous double haploids. Recent studies have shown that in vitro cultures are the origin of the so-called tissue culture-induced variation (TCIV) that may lead to off-type regenerants. Moreover, the regeneration of green plants may be limited by the presence of albinos. It was demonstrated that the presence of Cu2+ and Ag+ ions in the regeneration medium might increase the number of green plants.ResultsDArTseqMet markers were evaluated based on regenerants and donor plants derived via in vitro anther cultures of barley. The regenerants were obtained under varying Cu2+ and Ag+ ion concentration in the regeneration medium during distinct time conditions of the tissue cultures. The DArTseqMet markers were quantified using a semi-quantitative MSAP approach delivering data on CG and CHG sequence contexts de novo methylation and demethylation. Under each tissue culture conditions, the number of regenerated green plants per 100 anthers was evaluated. Conditional moderation analysis was applied to test for the role of Cu2+ and Ag+ ions in the medium. Moreover, the importance of the time of in vitro anther cultures were analyzed.ConclusionsOur data demonstrate that DNA de novo methylation and demethylation affecting CG and CXG DNA sequence contexts is moderated by the presence of Cu2+ and Ag+ ions in the medium conditional on the time of in vitro tissue cultures. The level of de novo methylation and demethylation and the difference between the two is essential for the understanding of moderation. Moreover, Cu2+ and Ag+ play in concert moderating DNA methylation changes. For the in vitro tissue culture purposes, the lower the delta value equal to de novo methylation less demethylation and the higher the value of the (Cu+Ag) predictor conditional on time, the higher the number of green plants should be evaluated. Moreover, evaluation of GPs is even more probable under positive delta and higher (Cu+Ag) values. Our data are congruent with the putative function of these ions in the ethylene and DNA methylation pathways.

scholarly journals Somatic embryogenesis from in vitro anther culture of apomictic buffel grass genotypes and analysis of regenerated plants using flow cytometry

2014 ◽  
Vol 117 (3) ◽  
pp. 311-322 ◽  
Author(s):  
Edgardo Carloni ◽  
Andrea Ribotta ◽  
Eliana López Colomba ◽  
Sabrina Griffa ◽  
Mariana Quiroga ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Somatic Embryogenesis ◽  
Anther Culture ◽  
Buffel Grass ◽  
Regenerated Plants

scholarly journals Genotypic features of morphogenetic reactions of varieties and hybrids F1 of winter soft wheat during various stages of androgenesis in vitro

2018 ◽  
Vol 22 ◽  
pp. 252-256 ◽  
Author(s):  
I. S. Zambriborshch ◽  
O. L. Shestopal ◽  
M. S. Boyko
Keyword(s):  
Winter Wheat ◽  
Anther Culture ◽  
Triticum Aestivum L ◽  
Soft Wheat ◽  
F1 Hybrids ◽  
Green Plants ◽  
Ability To Regenerate ◽  
Androgenesis In Vitro ◽  
Soft Winter Wheat

Aim. Testing the haploproduction ability of 47 varieties and 143 F1 hybrids of winter bread wheat. Methods. In vitro culture of isolated anthers of wheat. The percentage of callus and regeneration of green plants for each genotype calculated as a percentage of the planted anthers. Results. The differences in the frequency of сallus induction and the ability to regenerate plants in the process of androgenesiss in vitro of winter soft wheat were detected. The range of variation haploproduction activiti was broad. The sign of "the formation of сallus" for varieties was in limited from 0 to 21.2 % and for hybrids – from 0 to 38.8 %; on the sign of "regeneration of green plants" for varieties – from 0 to 9.4 %, and for hybrids from 0 to 9.1 %. The positive effect of 1BL/1RS translocation on signs of haploproduction in anther culture Triticum aestivum L. Conclusions. Genotype-specific of microspores morphogenetic reactions of soft winter wheat in the process of androgenesis in vitro were revealed. It has been found that F1 hybrids have a higher potential production potential in anther culture than varieties. During the period from 2011 to 2017, the 580 lines of winter wheat were transferred to the breeding departments. Keywords: soft winter wheat, anther culture in vitro, callus, regeneration.

Altered chromatin condensation of heat-stressed spermatozoa perturbs the dynamics of DNA methylation reprogramming in the paternal genome after in vitro fertilisation in cattle

2014 ◽  
Vol 26 (8) ◽  
pp. 1107 ◽  
Author(s):  
Mohammad Bozlur Rahman ◽  
Md. Mostofa Kamal ◽  
Tom Rijsselaere ◽  
Leen Vandaele ◽  
Mohammed Shamsuddin ◽  
...  
Keyword(s):  
Dna Methylation ◽  
De Novo ◽  
Chromatin Condensation ◽  
Dna Demethylation ◽  
In Vitro Fertilisation ◽  
Paternal Genome ◽  
Time Points ◽  
Fertilisation Rate ◽  
De Novo Methylation

Shortly after penetration of the oocyte, sperm DNA is actively demethylated, which is required for totipotent zygotic development. Aberrant DNA methylation is thought to be associated with altered chromatin condensation of spermatozoa. The objectives of this study were to investigate the dynamics of DNA methylation reprogramming in the paternal pronucleus and subsequent fertilisation potential of heat-stressed bull spermatozoa having altered chromatin condensation. Hence, bovine zygotes (n = 1239) were collected at three different time points (12, 18 and 24 h post insemination, hpi), and stained with an antibody against 5-methylcytosine. Fluorescence intensities of paternal and maternal pronuclei were measured by ImageJ. DNA methylation patterns in paternal pronuclei derived from heat-stressed spermatozoa did not differ between time points (P > 0.05), whereas control zygotes clearly showed demethylation and de novo methylation at 18 and 24 hpi, respectively. Moreover, heat-stressed spermatozoa showed a highly reduced (P < 0.01) fertilisation rate compared with non-heat-stressed or normal control spermatozoa (53.7% vs 70.2% or 81.5%, respectively). Our data show that the normal pattern of active DNA demethylation followed by de novo methylation in the paternal pronucleus is perturbed when oocytes are fertilised with heat-stressed spermatozoa, which may be responsible for decreased fertilisation potential.

Histology of organogenesis and somatic embryogenesis in excised root cultures of an endangered species Tylophora indica (Asclepiadaceae)

2010 ◽  
Vol 58 (3) ◽  
pp. 198 ◽  
Author(s):  
Aastha Sahai ◽  
Anwar Shahzad ◽  
Shiwali Sharma
Keyword(s):  
Somatic Embryogenesis ◽  
Induction Medium ◽  
Root Induction ◽  
Ms Medium ◽  
Tylophora Indica ◽  
Regenerated Plants ◽  
Maturation Medium ◽  
Half Strength ◽  
One Year

This paper reports an efficient regeneration protocol through parallel organogenic and embryogenic pathways from green root segments (GRSs) of Tylophora indica (Burm.f) Merrill. GRSs explants from one year old in vitro cultures were cultured on Murashige and Skoog (MS) medium containing various cytokinins. Five µmol/L of 6-benzyladenine (BA) was most responsive for organogenesis in 1.5 cm long GRSs. Repeated subculture on medium containing both BA (5 µmol/L) and 1-naphthleneacetic acid (NAA) (0.1 µmol/L) promoted multiplication and proliferation of direct shoot buds (46.80 ± 0.96) and callus mediated somatic embryogenesis (18.07 ± 0.33). Germinated embryos isolated from callus were transferred onto maturation medium consisting of half-strength MS medium either devoid of plant growth regulators (PGRs) or with various concentrations of gibberellic acid (GA). Microshoots were excised during subculture and transferred onto root induction medium, thus ensuring a continuous supply of germplasm. Morphogenic variations were noticed in types of roots induced on various auxins. Regenerated plantlets and emblings hardened best on vermiculite with a survival rate of 90% and 70% respectively. However, the emblings were healthier in comparison to the regenerated plants. Histological analysis showed the origin and development of organogenesis.

scholarly journals Utilisation of in vitro Techniques in Rescue of Gene Resources of Meadow Vetchling (Lathyrus pratensis L.)

2011 ◽  
Vol 39 (No. 3) ◽  
pp. 84-88 ◽  
Author(s):  
L. Klčová ◽  
M. Gubišová
Keyword(s):  
Culture Medium ◽  
Nodal Segments ◽  
Induction Medium ◽  
Ms Medium ◽  
In Vitro Techniques ◽  
In Vitro Methods ◽  
Regenerated Plants ◽  
Plant Maintenance

In the case of poor germination of seed samples and minimal number of seedlings obtained, in vitro methods can be used to revitalise and recover the gene resource. The highest germination of meadow vetchling (Lathyrus pratensis L.) seeds was achieved after scarification with H<sub>2</sub>SO<sub>4</sub> and cultivation in MS medium. The seedlings were used as a material for micropropagation. Regeneration passed through nodal segments cultivated on basal MS medium solidified with a combination of agar and phytagel. This culture medium was also suitable for the plant maintenance. An addition of cytokinin to the induction medium did not support multiplication and growth. In the basal MS medium rooted 72.5% (gene resource 62) or 42.5% (gene resource 28) of shoots. The rooting of gene resource 28 was increased to 63% by the addition of indolylbutyric acid to the culture medium. The regenerated plants were successfully transferred to the soil. This protocol can be used to rescue gene resources of this species. &nbsp; &nbsp;

scholarly journals The testing of haploproduction ability of soft winter wheat different hybrids in anther culture in vitro

2020 ◽  
Vol 26 ◽  
pp. 207-211
Author(s):  
I. S. Zambriborshch ◽  
O. L. Shestopal ◽  
M. S. Chekalova ◽  
E. A. Golub
Keyword(s):  
Winter Wheat ◽  
Anther Culture ◽  
Hybrid Population ◽  
Culture In Vitro ◽  
Green Plants ◽  
Ability To Regenerate ◽  
Androgenesis In Vitro ◽  
Soft Winter Wheat ◽  
Range Of Variation

Aim. Study the the efficiency of androgenesis in vitro in anther culture of different genotyps winter wheat. Methods. Obtaining of soft winter wheat double haploid lines by anther culture in vitro. The statistical methods. Results The ability to androgenesis in an in vitro anthers culture of 30 soft winter wheat genotypes was tested. The differences in the frequency of callus induction and the ability to regenerate plants in the process of androgenesiss in vitro of winter soft wheat were detected. The range of variation haploproduction activiti (from % of implanted anthers) was broad. The sign of "the formation of callus" was in limited from 0 to 13.2 % and on the sign of "regeneration of green plants" – from 0 to 1.8 %. The 126 green plants-regenerants were received. Conclusions. Genotype-specific of microspores morphogenetic reactions of soft winter wheat in the process of androgenesis in vitro were revealed. It is advisable to use the anthers from the F2 hybrid population plant as a donor material to create an effective technology for producing double haploids of soft winter wheat was shown. Keywords: wheat hybrid, anther culture in vitro, callus, regeneration.

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