scholarly journals Homozygous Transgenic Barley (Hordeum vulgare L.) Plants by Anther Culture

Plants ◽  
2020 ◽  
Vol 9 (7) ◽  
pp. 918
Author(s):  
Ludmila Ohnoutková ◽  
Tomáš Vlčko
Keyword(s):  
Anther Culture ◽  
Hordeum Vulgare L ◽  
Transgenic Barley ◽  
Embryo Cultures ◽  
Isolated Microspores ◽  
Transgenic Lines ◽  
Dh Lines ◽  
Transgenic Progeny ◽  
Selection Of

Production of homozygous lines derived from transgenic plants is one of the important steps for phenotyping and genotyping transgenic progeny. The selection of homozygous plants is a tedious process that can be significantly shortened by androgenesis, cultivation of anthers, or isolated microspores. Doubled haploid (DH) production achieves complete homozygosity in one generation. We obtained transgenic homozygous DH lines from six different transgenic events by using anther culture. Anthers were isolated from T0 transgenic primary regenerants and cultivated in vitro. The ploidy level was determined in green regenerants. At least half of the 2n green plants were transgenic, and their progeny were shown to carry the transgene. The process of dihaploidization did not affect the expression of the transgene. Embryo cultures were used to reduce the time to seed of the next generation. The application of these methods enables rapid evaluation of transgenic lines for gene function studies and trait evaluation.

scholarly journals Early in vitro selection of winter oilseed rape (Brassica napus L.) plants with the fertility restorer gene for CMS Shaan 2A via non-destructive molecular analysis of microspore-derived embryos

2019 ◽  
Vol 55 (No. 4) ◽  
pp. 162-165
Author(s):  
Miroslav Klíma ◽  
Eva Jozová ◽  
Irena Jelínková ◽  
Vratislav Kučera ◽  
Shengwu Hu ◽  
...  
Keyword(s):  
Brassica Napus ◽  
Oilseed Rape ◽  
In Vitro Selection ◽  
Restorer Gene ◽  
Brassica Napus L ◽  
Rf Gene ◽  
Dh Lines ◽  
Non Destructive ◽  
Selection Of

Cotyledons of 201 microspore-derived oilseed rape (Brassica napus) embryos were analysed for the presence of the cytoplasmic male sterility Shaan 2A restorer gene using the primer pair 3F/4R. In total, 172 plants regenerated and were grown to the generative stage. Among phenotypically non-haploid flowering plants, there was a broad spectrum of different flower types from normally developed flowers to flowers with short anthers, stamens, or deformed flowers with atypically developed floral organs, often with traces of pollen. For this reason, only 115 typically fertile or sterile accessions were selected for further phenotype-genotype comparisons. In 14 plants (12.2%) the phenotype did not match the genotype: two plants were marked as fertile, but without the Rf gene. Vice versa, in 12 sterile plants the Rf gene was determined. Nevertheless, the two fertile plants did not produce any pods after self-pollination. In total, 47 doubled haploid (DH) lines with good seed yield, carrying the Rf gene were harvested.  

scholarly journals Generation of doubled haploid lines from winter wheat (Triticum aestivum L.) breeding material using in vitro anther culture

2020 ◽  
Vol 56 (No. 4) ◽  
pp. 150-158
Author(s):  
Osama Zuhair Kanbar ◽  
Csaba Lantos ◽  
Paul Karumba Chege ◽  
Erzsébet Kiss ◽  
János Pauk
Keyword(s):  
Triticum Aestivum ◽  
Winter Wheat ◽  
Anther Culture ◽  
Doubled Haploid ◽  
Genetic Material ◽  
Triticum Aestivum L ◽  
Average Value ◽  
Doubled Haploid Lines ◽  
Dh Lines

We investigated the anther culture (AC) efficiency of thirteen F<sub>4</sub> combinations of winter wheat (Triticum aestivum L.). The genotype dependency was assessed during the induction of the androgenic entities, i.e. embryo-like structures (ELS), regenerated-, green-, albino-, and transplanted plantlets. The number of green plantlets per 100 anthers (GP/100A) varied from 0.36 to 24.74 GP/100A with a mean of 8.31 GP/100A. Albino plantlets (AP) occurred in each combination, ranging from 0.20 to 22.80 AP/100A with an average value of 5.59 AP/100A. Between 25–87.76 doubled haploid (DH) plants per 100 acclimatised plantlets (DH/100ADP), depending on the combination, with a mean of 59.74% were recovered. We have found the highest DH production in the combinations Béres/Midas, Kalász/Tacitus, Béres/Pamier, and Premio/5009. This improves remarkably the choice of basic genetic material in subsequent crossing programmes. These observations emphasise the usability and efficiency of in vitro AC in producing a large number of DH lines for breeding and the applied researches of winter wheat. Although albinism was found in each combination, it was mitigated by the in vitro AC application.

scholarly journals Utilization of in Vitro Anther Culture in Spelt Wheat Breeding

Plants ◽  
2019 ◽  
Vol 8 (10) ◽  
pp. 436 ◽  
Author(s):  
Csaba Lantos ◽  
Szandra Purgel ◽  
Katalin Ács ◽  
Bernadett Langó ◽  
Lajos Bóna ◽  
...  
Keyword(s):  
Anther Culture ◽  
Heading Date ◽  
Wheat Breeding ◽  
F1 Hybrids ◽  
Spelt Wheat ◽  
Advanced Line ◽  
Flour Yield ◽  
The Mean ◽  
Dh Lines

The efficiency of in vitro anther culture was screened in a full diallel population of four spelt wheat genotypes and ten F1 hybrids. Genotype dependency was observed based on the data of embryo-like structures (ELS), green-, albino plantlets. In the diallel population and ten F1 hybrids, the green plantlets production ranged from 13.75 to 85.00 and from 6.30 to 51.00, respectively. The anther culture-derived plants of F1 hybrids were grown up in the nursery. At the harvest, 436 spontaneous doubled haploid (DH) plants were identified among the 1535 anther culture-derived transplanted and grown up individual plants. The mean of spontaneous rediploidization was 28.4% which ranged from 9.76% to 54.24%. In two consecutive years, the agronomic values of ‘Tonkoly.pop1’ advanced line were compared with seven DH lines of ‘Tonkoly.pop1’ in the nursery. The DH lines achieved competitive values in comparison with ‘Tonkoly.pop1’ advanced line based on the 11 measured parameters (heading date, plant height, yield, hardness, width and length of seed, TKW, hulling yield, flour yield, protein and wet gluten content). These observations presage the efficient utilization of anther culture in spelt wheat breeding.

scholarly journals CULTURE USE PROBLEMS IN SELECTION OF ISOLATED MICROSPORES IN GRAIN

2020 ◽  
Vol 55 (1) ◽  
pp. 31-39
Author(s):  
G. A. Iskakova ◽  
D. O. Baisaparova ◽  
D. R. Raimbek ◽  
K. Zh. Zhambakin
Keyword(s):  
Genetic Basis ◽  
Selection Process ◽  
Microspore Culture ◽  
Wheat Breeding ◽  
Identification System ◽  
Cultivar Development ◽  
Isolated Microspores ◽  
New Gene ◽  
Selection Of

Production of haploid plants by culture of isolated microspores is a quick way of obtaining homozygous crop lines. Recessive features of mutant homozygous plants are also possible to determine by this biotechnology. Contrary from anthers culture, in which the presence of anther walls can lead to the development of diploid somatic calli and plants, the microspore culture produces only haploid or dihaploid lines. Isolated microspores culture in addition represents and has a unique identification system for studying the mechanisms of embryogenesis in in vitro culture. The usage of haploid technology extends the genetic basis of wheat breeding, since it allows increasing the frequency of new gene combinations. This technology significantly increases the efficiency of breeding new highly productive varieties of crops. On this basis, it becomes possible to quickly assess the prospects of dihaploids, which significantly improves the efficiency of the selection process. DH plants are completely fertile and, if necessary, may be used as parents or processed as a cultivar. DHs have been widely used for cultivar development, genetic mapping, mutagenesis, and the study of gene functions.

scholarly journals Developmental Sensitivity inSchistosoma mansonito Puromycin To Establish Drug Selection of Transgenic Schistosomes

2018 ◽  
Vol 62 (8) ◽  
Author(s):  
Hong-Bin Yan ◽  
Michael J. Smout ◽  
Chuan Ju ◽  
Anne E. Folley ◽  
Danielle E. Skinner ◽  
...  
Keyword(s):  
Developmental Stages ◽  
Helminth Parasites ◽  
Drug Selection ◽  
Loss Of Function ◽  
Transgenic Lines ◽  
Parasite Biology ◽  
Important Disease ◽  
Selection Of ◽  
Dual Staining

ABSTRACTSchistosomiasis is considered the most important disease caused by helminth parasites, in terms of morbidity and mortality. Tools to facilitate gain- and loss-of-function approaches can be expected to precipitate the discovery of novel interventions, and drug selection of transgenic schistosomes would facilitate the establishment of stable lines of engineered parasites. Sensitivity of developmental stages of schistosomes to the aminonucleoside antibiotic puromycin was investigated. For the schistosomulum and sporocyst stages, viability was quantified by fluorescence microscopy following dual staining with fluorescein diacetate and propidium iodine. By 6 days in culture, the 50% lethal concentration (LC50) for schistosomula was 19 μg/ml whereas the sporocysts were 45-fold more resilient. Puromycin potently inhibited the development ofin vitro-laid eggs (LC50, 68 ng/ml) but was less effective against liver eggs (LC50, 387 μg/ml). Toxicity for adult stages was evaluated using the xCELLigence-based, real-time motility assay (xWORM), which revealed LC50s after 48 h of 4.9 and 17.3 μg/ml for male and female schistosomes, respectively. Also, schistosomula transduced with pseudotyped retrovirus encoding the puromycin resistance marker were partially rescued when cultured in the presence of the antibiotic. Together, these findings will facilitate selection on puromycin of transgenic schistosomes and the enrichment of cultures of transgenic eggs and sporocysts to facilitate the establishment of schistosome transgenic lines. Streamlining schistosome transgenesis with drug selection will open new avenues to understand parasite biology and hopefully lead to new interventions for this neglected tropical disease.

scholarly journals Overexpression of Nepenthesin HvNEP-1 in Barley Endosperm Reduces Fusarium Head Blight and Mycotoxin Accumulation

Agronomy ◽  
2020 ◽  
Vol 10 (2) ◽  
pp. 203 ◽  
Author(s):  
Zelalem Eshetu Bekalu ◽  
Claus Krogh Madsen ◽  
Giuseppe Dionisio ◽  
Inger Bæksted Holme ◽  
Lise Nistrup Jørgensen ◽  
...  
Keyword(s):  
Fusarium Head Blight ◽  
Genetic Resistance ◽  
Fusarium Culmorum ◽  
Substantial Reduction ◽  
Field Evaluation ◽  
Head Blight ◽  
Hordeum Vulgare L ◽  
Transgenic Barley ◽  
Transgenic Lines ◽  
Barley Grains

Fusarium head blight (FHB) causes substantial losses of yield and quality in grains, both in the field and in post-harvest storage. To date, adequate natural genetic resistance is not available for the control of FHB. This study reports the cloning and overexpression of a barley (Hordeum vulgare L.) antifungal gene, nepenthesin 1 (HvNEP-1), in the endosperm of barley grains. Transgenic barley lines overexpressing HvNEP-1 substantially reduced FHB severity and disease progression after inoculation with Fusarium graminearum or Fusarium culmorum. The transgenic barley also showed reduced accumulation of the mycotoxin deoxynivalenol (DON) in grain, far below the minimum value allowable for food. Semi-field evaluation of four HvNEP-1 transgenic lines revealed substantial reduction of FHB severity and progression as compared with the control H. vulgare cultivar Golden promise (GP) plants. Our study demonstrated the utility of HvNEP-1 for the control of FHB in barley, and possibly other grains such as wheat and maize.

scholarly journals Androgenic responses of winter wheat (Triticum aestivum L.) combinations in in vitro anther culture

Genetika ◽  
2020 ◽  
Vol 52 (1) ◽  
pp. 335-350
Author(s):  
Osama Kanbar ◽  
Csaba Lantos ◽  
Erzsebet Kiss ◽  
Janos Pauk
Keyword(s):  
Winter Wheat ◽  
Anther Culture ◽  
Triticum Aestivum L ◽  
Wheat Breeding ◽  
Published Data ◽  
In Vitro Production ◽  
The Mean ◽  
Dh Lines ◽  
Vitro Production

The androgenic parameters were investigated by in vitro anther culture (AC) on nine F2-5 breeding combinations of winter wheat (Triticum aestivium L.). Each combination produced embryo-like structures (ELS), green plantlets, albino plantlets, transplanted plantlets and acclimatized plantlets, with respect to the number of anthers in AC. The number of AC-derived ELS was between 11.73 and 52.76 ELS/100 anthers with the mean of 26.22 ELS/100 anthers, out of which the number of regenerated green plantlets varied from 3.20 to 26.40 green plantlets/100 anthers and the mean was 9.76 green plantlets/100 anthers, while the number of transplanted plantlets ranged from 2.16 to 21.77 transplanted plantlets/100 anthers. Furthermore, the number of albinos/100 anthers was mitigated and varied between 0.72 and 6.20 albinos/100 anthers. We also studied the number of green and albino plantlets per 100 ELS. The rate of green plantlets per 100 ELS ranged between 14.81% and 64.01%, with the overall mean 33.59%, while the rate of albinos per 100 ELS ranged from 4.82% to 20.70% with the overall mean 11.93%. In our experiment, the rate of acclimatized plantlets (70.15-91.57%) depended mostly on the combination. This study asserted the importance of AC method in wheat for in vitro production of green plants. Although albinism was found in each combination, it did not hinder the production of green plantlets. The satisfying results were achieved in green plantlets production compared to the previously published data, but further improvement will be needed continuously, experiment by experiment. The generated acclimatized plantlets will be used in the wheat breeding program as doubled haploid (DH) lines.

scholarly journals High production of wheat double haploids via anther culture

Genetika ◽  
2007 ◽  
Vol 39 (3) ◽  
pp. 405-411
Author(s):  
Ankica Kondic-Sipka ◽  
Boris Kobiljski ◽  
Nikola Hristov
Keyword(s):  
Anther Culture ◽  
Triticum Aestivum L ◽  
Green Plant ◽  
F1 Hybrids ◽  
Regeneration Ability ◽  
Breeding Programs ◽  
High Production ◽  
Dh Lines ◽  
Wheat Lines

Androgenous and regeneration abilities of 14 randomly selected F1 hybrids of wheat (Triticum aestivum L.) were analyzed. Anthers were grown in vitro on a modified Potato-2 inductive medium. The hybrid NS111-95/Ana had the highest average values for androgenous capacity (33%) and callus yield (119%), while the hybrid NS 92-250/Tiha had the lowest values for these traits (9 and 21%, respectively). Seven genotypes (50%) had a frequency of green plants relative to the number of isolated anthers of over 10%, with the highest frequency of 21.3% (NS111-95/Sremica). This hybrid produced 12.8 doubled haploid (DH) lines per spike used for isolation. In the other genotypes, the number of produced DH lines per spike ranged from 1 (30?Sc.Smoc.88-89/Hays-2) to 11.2 (NS111-95/Ana). As half of the randomly selected genotypes exhibited high green plant regeneration ability and a high production of DH lines per spike, it can be concluded that in vitro anther culture can be successfully used in breeding programs for rapid production of homozygous wheat lines.

scholarly journals Effect of trophic, osmotically active and solidifying components of nutrient medium on the direct embryognesis in spring barley anther culture in vitro

2018 ◽  
Vol 22 ◽  
pp. 210-215
Author(s):  
E. V. Belinskaya ◽  
P. G. Dulnyev
Keyword(s):  
Plant Regeneration ◽  
Anther Culture ◽  
Spring Barley ◽  
Gelling Agent ◽  
Modified Starch ◽  
Hordeum Vulgare L ◽  
Culture In Vitro ◽  
Chemically Modified ◽  
Determine Mechanism

Aim. Ambryoidogenesis, or somatic embryogenesis, is known to be the most efficient mode of plant regeneration in plant cell, tissue and organ culture. The investigation was aimed to elucidate effects of trophic and osmogenic components of inductive medium on the frequency of direct embryoidogenesis in spring barley anther culture in vitro and to determine mechanism of morphogenesis improvement coursed by chemically modified starch D-5aM used as a gelling agent instead of agar. Methods. Anthers of DH-line with a high androgenetic capacity were inoculated on inductive media containing N6 macro-, MS micronutrients, organic supplements, maltose or mannitol (0.3 M) and solidified with agar or chemically modified starch. Results. A positive effect of combination of high maltose content and chemically modified starch on the induction and regeneration processes in spring barley anther culture in vitro was confirmed. It was also shown that mannitol didn’t keep any growth or development processes going in barley anther and embryo culture, but at the same time this substance had no toxic effect. Conclusions. In order to achieve a high frequency of induction in spring barley anther culture, it is necessary to use medium containing maltose – a low weight component both with trophic and with osmotic activity. Keywords: Hordeum vulgare L., anther culture in vitro, mannitol, maltose, embryo formation, plant regeneration.

scholarly journals Response of Anther Donor Genotypes (F1) from Indica x Indica Crosses to Rice Anther Culture

2019 ◽  
Vol 15 (1) ◽  
pp. 45
Author(s):  
Iswari Saraswati Dewi ◽  
Nuha Hera Putri ◽  
Bambang Sapta Purwoko
Keyword(s):  
Anther Culture ◽  
First Generation ◽  
Development Program ◽  
Rice Cultivar ◽  
Similar Response ◽  
Donor Plant ◽  
In Vitro Techniques ◽  
Cultivar Development ◽  
Dh Lines

<p>Anther culture is one of the in vitro techniques that can be used to accelerate the obtainment of purelines in the form of dihaploid (DH) plants resulted from androgenesis. The objectives of this research were to study response of anther donor genotypes in rice anther culture of F1 populations derived from indica x indica crosses and obtain spontaneous DH lines to be used further for variety development program. Anther donor plant used for the experiment consisted of four kinds of F1 populations derived from single crosses, i.e. IR85/I-5, BioR-81/I-5, Bio-R81/O18, and BioR-82/O-18. The result indicated that kindy of all F1 plants showed similar response to callus induction, but significatly different responses to plant regeneration. Genotypes of anther donor plants (F1) significantly affected the percentage of calli producing green plantlet, but not significantly affected the percentage of calli producing albino plantlet. Anther culturability of anther donor plants (F1) was relatively high, ranged from 2.0 to 7.0%. A total of 73 first generation of spontaneous DH lines (38.8%) from the grew out green plantlets was obtained from this study. The resulted DH lines are readily available to be selected and be used in rice cultivar development program.</p>

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