scholarly journals Changes in plastid biogenesis leading to the formation of albino regenerants in barley microspore culture

2020 ◽  
Author(s):  
Monika Gajecka ◽  
Marek Marzec ◽  
Beata Chmielewska ◽  
Janusz Jelonek ◽  
Justyna Zbieszczyk ◽  
...  
Keyword(s):  
Microspore Culture ◽  
Doubled Haploids ◽  
Microspore Embryogenesis ◽  
Rrna Genes ◽  
23S Rrna ◽  
Gene Encoding ◽  
Plastid Differentiation ◽  
Barley Cultivars ◽  
Albino Plants ◽  
Genes Encoding

Abstract Background: Microspore embryogenesis is potentially the most effective method of obtaining doubled haploids (DH), which are utilized in breeding programs to accelerate production of new cultivars. However, the regeneration of albino plants significantly limits the exploitation of androgenesis for DH production in cereals. Despite many efforts, the precise mechanisms leading to development of albino regenerants have not yet been elucidated. The objective of this study was to reveal the genotype-dependent molecular differences in chloroplast differentiation that lead to the formation of green and albino regenerants in microspore culture of barley.Results: We performed a detailed analysis of plastid differentiation at successive stages of androgenesis in two barley cultivars, ‘Jersey’ and ‘Mercada’ that differed in their ability to produce green regenerants. We demonstrated the lack of transition from the NEP-dependent to PEP-dependent transcription in plastids of ‘Mercada’ that produced mostly albino regenerants in microspore culture. The failed NEP-to-PEP transition was associated with the lack of activity of Sig2 gene encoding a sigma factor necessary for transcription of plastid rRNA genes. The impaired PEP activity caused a very low level of 16S and 23S rRNA transcripts, lack of plastid translation machinery and inhibition of photomorphogenesis in regenerating embryos and albino regenerants. Furthermore, the plastids present in differentiating ‘Mercada’ embryos contained a low number of plastome copies whose replication was not always completed. Contrary to ‘Mercada’, ‘Jersey’ that produced 90% green regenerants, showed the high activity of PEP, the highly increased expression of Sig2, plastid rRNA and tRNAGlu transcripts, which indicated the NEP inhibition. The increased expression of GLKs genes encoding transcription factors required for induction of photomorphogenesis was also observed in ‘Jersey’ regenerants. Conclusions: Proplastids present in microspore-derived embryos of albino-producing genotypes did not pass the early checkpoint of their development that are required for induction of further light-dependent differentiation of chloroplasts. The failed activation of plastid-encoded RNA polymerase during differentiation of embryos was the main cause of the genotype-dependent inability to regenerate green plants in barley microspore culture. The better understanding of molecular mechanism underlying formation of albino regenerants may be helpful in overcoming the problem of albinism in cereal androgenesis.

scholarly journals Changes in plastid biogenesis leading to the formation of albino regenerants in barley microspore culture

2020 ◽  
Author(s):  
Monika Gajecka ◽  
Marek Marzec ◽  
Beata Chmielewska ◽  
Janusz Jelonek ◽  
Justyna Zbieszczyk ◽  
...  
Keyword(s):  
Microspore Culture ◽  
Doubled Haploids ◽  
Microspore Embryogenesis ◽  
Rrna Genes ◽  
23S Rrna ◽  
Gene Encoding ◽  
Plastid Differentiation ◽  
Barley Cultivars ◽  
Albino Plants ◽  
Genes Encoding

Abstract Background: Microspore embryogenesis is potentially the most effective method of obtaining doubled haploids (DH) which are utilized in breeding programs to accelerate production of new cultivars. However, the regeneration of albino plants significantly limits the exploitation of androgenesis for DH production in cereals. Despite many efforts, the precise mechanisms leading to development of albino regenerants have not yet been elucidated. The objective of this study was to reveal the genotype-dependent molecular differences in chloroplast differentiation that lead to the formation of green and albino regenerants in microspore culture of barley.Results: We performed a detailed analysis of plastid differentiation at successive stages of androgenesis in two barley cultivars, ‘Jersey’ and ‘Mercada’ that differed in their ability to produce green regenerants. We demonstrated the lack of transition from the NEP-dependent to PEP-dependent transcription in plastids of cv. ‘Mercada’ that produced mostly albino regenerants in microspore culture. The failed NEP-to-PEP transition was associated with the lack of activity of Sig2 gene encoding a sigma factor necessary for transcription of plastid rRNA genes. A very low level of 16S and 23S rRNA transcripts and impaired plastid translation machinery resulted in the inhibition of photomorphogenesis in regenerating embryos and albino regenerants. Furthermore, the plastids present in differentiating ‘Mercada’ embryos contained a low number of plastome copies whose replication was not always completed. Contrary to ‘Mercada’, cv. ‘Jersey’ that produced 90% green regenerants, showed the high activity of PEP polymerase, the highly increased expression of Sig2, plastid rRNAs and tRNAGlu, which indicated the NEP inhibition. The increased expression of GLKs genes encoding transcription factors required for induction of photomorphogenesis was also observed in ‘Jersey’ regenerants. Conclusions: Proplastids present in microspore-derived embryos of albino-producing genotypes did not pass the early checkpoint of their development that are required for induction of further light-dependent differentiation of chloroplasts. The failed activation of plastid-encoded RNA polymerase during differentiation of embryos was associated with the genotype-dependent inability to regenerate green plants in barley microspore culture. The better understanding of molecular mechanism underlying formation of albino regenerants may be helpful in overcoming the problem of albinism in cereal androgenesis.

scholarly journals Changes in plastid biogenesis leading to the formation of albino regenerants in barley microspore culture 

2020 ◽  
Author(s):  
Monika Gajecka ◽  
Marek Marzec ◽  
Beata Chmielewska ◽  
Janusz Jelonek ◽  
Justyna Zbieszczyk ◽  
...  
Keyword(s):  
Microspore Culture ◽  
Doubled Haploids ◽  
Microspore Embryogenesis ◽  
Rrna Genes ◽  
23S Rrna ◽  
Gene Encoding ◽  
Plastid Differentiation ◽  
Barley Cultivars ◽  
Albino Plants ◽  
Genes Encoding

Abstract Background: Microspore embryogenesis is potentially the most effective method of obtaining doubled haploids (DH) which are utilized in breeding programs to accelerate production of new cultivars. However, the regeneration of albino plants significantly limits the exploitation of androgenesis for DH production in cereals. Despite many efforts, the precise mechanisms leading to development of albino regenerants have not yet been elucidated. The objective of this study was to reveal the genotype-dependent molecular differences in chloroplast differentiation that lead to the formation of green and albino regenerants in microspore culture of barley.Results: We performed a detailed analysis of plastid differentiation at successive stages of androgenesis in two barley cultivars, ‘Jersey’ and ‘Mercada’ that differed in their ability to produce green regenerants. We demonstrated the lack of transition from the NEP-dependent to PEP-dependent transcription in plastids of cv. ‘Mercada’ that produced mostly albino regenerants in microspore culture. The failed NEP-to-PEP transition was associated with the lack of activity of Sig2 gene encoding a sigma factor necessary for transcription of plastid rRNA genes. A very low level of 16S and 23S rRNA transcripts and impaired plastid translation machinery resulted in the inhibition of photomorphogenesis in regenerating embryos and albino regenerants. Furthermore, the plastids present in differentiating ‘Mercada’ embryos contained a low number of plastome copies whose replication was not always completed. Contrary to ‘Mercada’, cv. ‘Jersey’ that produced 90% green regenerants, showed the high activity of PEP polymerase, the highly increased expression of Sig2, plastid rRNAs and tRNAGlu, which indicated the NEP inhibition. The increased expression of GLKs genes encoding transcription factors required for induction of photomorphogenesis was also observed in ‘Jersey’ regenerants. Conclusions: Proplastids present in microspore-derived embryos of albino-producing genotypes did not pass the early checkpoint of their development that are required for induction of further light-dependent differentiation of chloroplasts. The failed activation of plastid-encoded RNA polymerase during differentiation of embryos was associated with the genotype-dependent inability to regenerate green plants in barley microspore culture. The better understanding of molecular mechanism underlying formation of albino regenerants may be helpful in overcoming the problem of albinism in cereal androgenesis.

scholarly journals Changes in plastid biogenesis leading to the formation of albino regenerants in barley microspore culture

2021 ◽  
Vol 21 (1) ◽  
Author(s):  
Monika Gajecka ◽  
Marek Marzec ◽  
Beata Chmielewska ◽  
Janusz Jelonek ◽  
Justyna Zbieszczyk ◽  
...  
Keyword(s):  
Molecular Mechanisms ◽  
Microspore Culture ◽  
Doubled Haploids ◽  
Microspore Embryogenesis ◽  
Rrna Genes ◽  
23S Rrna ◽  
Gene Encoding ◽  
Plastid Differentiation ◽  
Barley Cultivars ◽  
Albino Plants

Abstract Background Microspore embryogenesis is potentially the most effective method of obtaining doubled haploids (DH) which are utilized in breeding programs to accelerate production of new cultivars. However, the regeneration of albino plants significantly limits the exploitation of androgenesis for DH production in cereals. Despite many efforts, the precise mechanisms leading to development of albino regenerants have not yet been elucidated. The objective of this study was to reveal the genotype-dependent molecular differences in chloroplast differentiation that lead to the formation of green and albino regenerants in microspore culture of barley. Results We performed a detailed analysis of plastid differentiation at successive stages of androgenesis in two barley cultivars, ‘Jersey’ and ‘Mercada’ that differed in their ability to produce green regenerants. We demonstrated the lack of transition from the NEP-dependent to PEP-dependent transcription in plastids of cv. ‘Mercada’ that produced mostly albino regenerants in microspore culture. The failed NEP-to-PEP transition was associated with the lack of activity of Sig2 gene encoding a sigma factor necessary for transcription of plastid rRNA genes. A very low level of 16S and 23S rRNA transcripts and impaired plastid translation machinery resulted in the inhibition of photomorphogenesis in regenerating embryos and albino regenerants. Furthermore, the plastids present in differentiating ‘Mercada’ embryos contained a low number of plastome copies whose replication was not always completed. Contrary to ‘Mercada’, cv. ‘Jersey’ that produced 90% green regenerants, showed the high activity of PEP polymerase, the highly increased expression of Sig2, plastid rRNAs and tRNAGlu, which indicated the NEP inhibition. The increased expression of GLKs genes encoding transcription factors required for induction of photomorphogenesis was also observed in ‘Jersey’ regenerants. Conclusions Proplastids present in microspore-derived embryos of albino-producing genotypes did not pass the early checkpoints of their development that are required for induction of further light-dependent differentiation of chloroplasts. The failed activation of plastid-encoded RNA polymerase during differentiation of embryos was associated with the genotype-dependent inability to regenerate green plants in barley microspore culture. The better understanding of molecular mechanisms underlying formation of albino regenerants may be helpful in overcoming the problem of albinism in cereal androgenesis.

The genetics of 5S rRNA encoding multigene families in barley

Genome ◽  
1993 ◽  
Vol 36 (6) ◽  
pp. 1023-1028 ◽  
Author(s):  
Vladimir Kanazin ◽  
Evgeny Ananiev ◽  
Tom Blake
Keyword(s):  
Tandem Repeats ◽  
Doubled Haploids ◽  
Gene Clusters ◽  
Variable Number ◽  
5S Rrna ◽  
Rrna Genes ◽  
Multigene Families ◽  
Obvious Effect ◽  
Barley Cultivars ◽  
5S Rrna Genes

Two loci containing genes encoding 5S rRNA were mapped on the second and third chromosomes of barley. The two gene clusters located on different chromosomes differed in the length of the nontranscribed spacer separating the 5S rRNA genes. All nontranscribed spacers contained a variable number of trinucleotide tandem repeats. The distribution of 5S genes between these two clusters and their copy number varied widely between cultivars and doubled haploids derived from a cross between two barley cultivars. However, this variation had no obvious effect on plant phenotype.Key words: 5S rRNA genes, multigene families, nontranscribed spacers, trinucleotide tandem repeats, barley, phenotype.

Study of androgenesis and spontaneous chromosome doubling in barley ( Hordeum vulgare L.) genotypes using isolated microspore culture

2009 ◽  
Vol 57 (2) ◽  
pp. 155-164 ◽  
Author(s):  
D. Kahrizi ◽  
R. Mohammadi
Keyword(s):  
Chromosome Doubling ◽  
Microspore Culture ◽  
Doubled Haploids ◽  
Negative Relationship ◽  
Microspore Embryogenesis ◽  
Culture Technique ◽  
Hordeum Vulgare L ◽  
Spontaneous Chromosome ◽  
Barley Genotypes

This research aimed to study the androgenesis and spontaneous chromosome doubling of five barley genotypes using an isolated in vitro microspore culture technique, involving a completely randomized design (CRD) with three replications. Statistical analysis of embryogenesis and cytogenetic results showed that genotype had a significant effect on haploid embryogenesis (P<0.01) and on spontaneous chromosome doubling (P<0.05). The genotype Igri was found to have the highest potential to produce haploid embryos (1577 embryos from 100 anthers), followed by the genotypes Boyer/Rojo, Afzal/Turkman/Kavir, Ashar/Hebo and Agrigashar/Matico with 369, 304, 278 and 150 embryos from 100 anthers, respectively. The highest percentage of spontaneous chromosome doubling (76%) was observed for the genotype which had the lowest embryogenesis (Agrigashar/Matico) and the lowest (65%) for the genotype with the highest androgenic capacity (Igri). Microspore embryogenesis also showed comparatively higher genotypic (109.2) and phenotypic (109.5) coefficients of variation, heritability (99.62) and genetic advance (1206.77), indicating the pre-dominance of additive gene action in the control of this character in the material studied. Estimates of genetic parameters (PCV, GCV and heritability) for microspore embryogenesis were higher than for spontaneous doubled haploids. These results indicated that selection for androgenic capacity would be more effective than for spontaneous doubled haploids. The findings showed a negative relationship (r= −0.68) between embryogenesis and spontaneous chromosome doubling in the barley genotypes studied. All the large embryos used had high regenerability and good plantlet formation.

scholarly journals Effects of Genotype and Culture Conditions on Microspore Embryogenesis and Plant Regeneration in Brassica Rapa ssp. Rapa L.

Plants ◽  
2020 ◽  
Vol 9 (2) ◽  
pp. 278 ◽  
Author(s):  
Daria Shumilina ◽  
Dmitry Kornyukhin ◽  
Elena Domblides ◽  
Alexey Soldatenko ◽  
Anna Artemyeva
Keyword(s):  
Microspore Culture ◽  
Doubled Haploids ◽  
Genetic Disorder ◽  
Cold Treatment ◽  
Microspore Embryogenesis ◽  
Culture Conditions ◽  
Secondary Embryogenesis ◽  
Isolated Microspore Culture ◽  
Pure Lines

Turnip is a biennial crop and, consequently, the creation of pure lines for breeding is a time-consuming process. The production of pure turnip lines using doubled haploids produced in isolated microspore culture has not been sufficiently developed. The aim of the present work was to determine some key factors inducing embryogenesis in the isolated microspore culture of turnip, as well as investigating the manners of embryo development. It was shown that the acidity of the medium is an important factor in embryo production; different optimal pH levels ranging from 6.2 to 6.6 corresponded to individual genotypes. Such factors as the cold treatment of buds and the addition of activated charcoal to the nutrient medium increased the responsiveness of all genotypes studied. The turnip variety ‘Ronde witte roodkop herfst’ demonstrated a genetic disorder in the development of microspores; namely, non-separation of some microspores from tetrads. In the in vitro culture, each of the daughter microspores developed on its own. This indicates the dependence of the possibility of embryogenesis in the turnip microspore culture on the genotype. Results suggest that the initiation of secondary embryogenesis in primary embryos leads to an increase in the proportion of doubled haploid plants.

scholarly journals Antibiotic Susceptibility and Sequence Type Distribution of Ureaplasma Species Isolated from Genital Samples in Switzerland

2015 ◽  
Vol 59 (10) ◽  
pp. 6026-6031 ◽  
Author(s):  
Sarah C. Schneider ◽  
Regula Tinguely ◽  
Sara Droz ◽  
Markus Hilty ◽  
Valentina Donà ◽  
...  
Keyword(s):  
Ribosomal Proteins ◽  
Ureaplasma Urealyticum ◽  
Mycoplasma Hominis ◽  
16S Rrna Genes ◽  
Rrna Genes ◽  
23S Rrna ◽  
Content Type ◽  
Ureaplasma Parvum ◽  
Genes Encoding ◽  
Antimicrobial Susceptibility Tests

ABSTRACTAntibiotic resistance inUreaplasma urealyticum/Ureaplasma parvumandMycoplasma hominisis an issue of increasing importance. However, data regarding the susceptibility and, more importantly, the clonality of these organisms are limited. We analyzed 140 genital samples obtained in Bern, Switzerland, in 2014. Identification and antimicrobial susceptibility tests were performed by using the Mycoplasma IST 2 kit and sequencing of 16S rRNA genes. MICs for ciprofloxacin and azithromycin were obtained in broth microdilution assays. Clonality was analyzed with PCR-based subtyping and multilocus sequence typing (MLST), whereas quinolone resistance and macrolide resistance were studied by sequencinggyrA, gyrB,parC, andparEgenes, as well as 23S rRNA genes and genes encoding L4/L22 ribosomal proteins. A total of 103 samples were confirmed as positive forU. urealyticum/U. parvum, whereas 21 were positive for bothU. urealyticum/U. parvumandM. hominis. According to the IST 2 kit, the rates of nonsusceptibility were highest for ciprofloxacin (19.4%) and ofloxacin (9.7%), whereas low rates were observed for clarithromycin (4.9%), erythromycin (1.9%), and azithromycin (1%). However, inconsistent results between microdilution and IST 2 kit assays were recorded. Various sequence types (STs) observed previously in China (ST1, ST2, ST4, ST9, ST22, and ST47), as well as eight novel lineages, were detected. Only some quinolone-resistant isolates had amino acid substitutions in ParC (Ser83Leu inU. parvumof serovar 6) and ParE (Val417Thr inU. parvumof serovar 1 and the novel Thr417Val substitution inU. urealyticum). Isolates with mutations in 23S rRNA or substitutions in L4/L22 were not detected. This is the first study analyzing the susceptibility ofU. urealyticum/U. parvumisolates in Switzerland and the clonality outside China. Resistance rates were low compared to those in other countries. We hypothesize that some hyperepidemic STs spread worldwide via sexual intercourse. Large combined microbiological and clinical studies should address this important issue.

scholarly journals The effect of copper on plant regeneration in barley microspore culture

2017 ◽  
Vol 53 (No. 1) ◽  
pp. 17-22 ◽  
Author(s):  
K. Makowska ◽  
S. Oleszczuk ◽  
J. Zimny
Keyword(s):  
Plant Regeneration ◽  
Copper Sulphate ◽  
Microspore Culture ◽  
Doubled Haploids ◽  
Spring Barley ◽  
Culture Method ◽  
Induction Medium ◽  
Isolated Microspore Culture ◽  
Albino Plants ◽  
Effect Of Copper

Isolated microspore culture is an excellent system for the production of doubled haploids in many crops, including barley. In a more traditional barley anther culture method copper sulphate is known to enhance plant regeneration. Here we report that one hundred times higher concentration of copper sulphate in the isolated microspore culture of two spring barley genotypes compared to the standard content in the induction medium resulted in a 34% increase of total plant regeneration. Detailed analysis of plant regeneration showed that additional supplementation of copper sulphate increased not only the regeneration of green plants but also proportionately that of albino plants. Hence, the results from two studied genotypes do not support an assumption that the addition of copper reduces albinism in barley microspore culture.

scholarly journals cfr(B), cfr(C), and a New cfr-Like Gene, cfr(E), in Clostridium difficile Strains Recovered across Latin America

2019 ◽  
Vol 64 (1) ◽  
Author(s):  
Vanja Stojković ◽  
María Fernanda Ulate ◽  
Fanny Hidalgo-Villeda ◽  
Emmanuel Aguilar ◽  
Camilo Monge-Cascante ◽  
...  
Keyword(s):  
Latin America ◽  
Clostridium Difficile ◽  
Ribosomal Proteins ◽  
Rrna Genes ◽  
23S Rrna ◽  
Cross Resistance ◽  
Content Type ◽  
Integrative And Conjugative Elements ◽  
Genes Encoding

ABSTRACT Cfr is a radical S-adenosyl-l-methionine (SAM) enzyme that confers cross-resistance to antibiotics targeting the 23S rRNA through hypermethylation of nucleotide A2503. Three cfr-like genes implicated in antibiotic resistance have been described, two of which, cfr(B) and cfr(C), have been sporadically detected in Clostridium difficile. However, the methylase activity of Cfr(C) has not been confirmed. We found cfr(B), cfr(C), and a cfr-like gene that shows only 51 to 58% protein sequence identity to Cfr and Cfr-like enzymes in clinical C. difficile isolates recovered across nearly a decade in Mexico, Honduras, Costa Rica, and Chile. This new resistance gene was termed cfr(E). In agreement with the anticipated function of the cfr-like genes detected, all isolates exhibited high MIC values for several ribosome-targeting antibiotics. In addition, in vitro assays confirmed that Cfr(C) and Cfr(E) methylate Escherichia coli and, to a lesser extent, C. difficile 23S rRNA fragments at the expected positions. The analyzed isolates do not have mutations in 23S rRNA genes or genes encoding the ribosomal proteins L3 and L4 and lack poxtA, optrA, and pleuromutilin resistance genes. Moreover, these cfr-like genes were found in Tn6218-like transposons or integrative and conjugative elements (ICE) that could facilitate their transfer. These results indicate selection of potentially mobile cfr-like genes in C. difficile from Latin America and provide the first assessment of the methylation activity of Cfr(C) and Cfr(E), which belong to a cluster of Cfr-like proteins that does not include the functionally characterized enzymes Cfr, Cfr(B), and Cfr(D).

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