scholarly journals The Influence of pH on Microspore Embryogenesis of White Cabbage (Brassica oleracea L.)

2013 ◽  
Vol 5 (4) ◽  
pp. 485-489 ◽  
Author(s):  
Tina Oana CRISTEA
Keyword(s):  
Brassica Oleracea ◽  
Microspore Culture ◽  
Microspore Embryogenesis ◽  
Critical Factor ◽  
Cellular Level ◽  
Strong Base ◽  
White Cabbage ◽  
In Vitro Microspore Culture ◽  
Influence Of Ph

In vitro microspore culture is one of the top techniques utilised now-a-days for the obtaining of double haploid plants in many plant species, including Brassica. The pH of the medium is a critical factor for the success of In vitro microspore culture as it influences the invertase enzyme activity, translated at cellular level through an acceleration or reduction of sucrose cleavage. The results published until now shows rather contradictory findings, as the response of microspores have been proved to be highly depending on genotypes, most of them being focused on Brassica napus. Thus, in the present study, the effect of different NLN liquid medium pH, ranging between 5.0 to 7.0 were tested in order to establish the most suitable pH for the expression of embryogenic competences of microspores cultivated on medium In vitro and ultimately for the obtaining of microspore-derived embryos. Among the 11 values of pH tested, the best results were obtained on variants with pH 5.8 and 6.0, both in what concern the maintaining of microspores viability and the number of microspore-derived embryos. The findings of the present study provide a strong base for the establishment of an efficient protocol for the In vitro culture of microspore at Brassica oleracea L. genotypes with Romanian origin.

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 Improvements of doubled haploid production protocol for white cabbage (Brassica oleracea var. capitata L.)

2018 ◽  
Vol 30 (1) ◽  
pp. 57-66 ◽  
Author(s):  
Katarina Rudolf Pilih ◽  
Urška Karolina Potokar ◽  
Borut Bohanec
Keyword(s):  
Brassica Oleracea ◽  
Doubled Haploid ◽  
Microspore Culture ◽  
Petri Dish ◽  
Abnormal Development ◽  
Haploid Induction ◽  
Embryo Formation ◽  
White Cabbage ◽  
Dh Lines ◽  
Further Development

Abstract Protocols leading to the development of doubled haploid (DH) lines by microspore culture are widely used in white cabbage (Brassica oleracea var. capitata L.), but efficiency varies according to the cultivar and induction procedure. Forty different genotypes consisting of F1 cultivars and their crosses with responsive doubled haploid lines were tested to evaluate the androgenic response. In total, 20,032 embryos were produced. On average, the haploid induction response of F1 cultivars was 7.0 embryos/Petri dish, but the average of these hybrids crossed to responsive DH lines was 26.6 embryos/Petri dish. In seven reciprocal crosses, a difference was observed in just one, meaning that the maternal effect probably has a minor influence on haploid embryogenesis in cabbage. Addition of 0.02% activated charcoal (AC) to the induction media increased embryo formation in several low-responsive genotypes, but its effect on embryo formation of high-responsive genotypes was predominantly negative, although larger embryos were formed on media containing AC than without AC. Further development into plantlets was tested by two procedures. Formed embryos were either transferred directly to regeneration medium or treated with abscisic acid and desiccated for 4 weeks. Regrowth and further development reached on average 15.5 and 57.6%, for the first and second procedures, respectively. Plantlets developed by direct transfer often exhibited abnormal development or hyperhydricity, unlike the desiccated embryos. Spontaneous diploidisation of embryos reached 42.5% in total and was not affected by AC added to the induction media.

scholarly journals In situ detection of Esr proteins secretion during maize microspore embryogenesis and their secretion blockage show effects on the culture progression

2010 ◽  
Vol 37 (10) ◽  
pp. 985 ◽  
Author(s):  
Pilar S. Testillano ◽  
María-José Coronado ◽  
Anne-Marie Thierry ◽  
Elisabeth Matthys-Rochon ◽  
María C. Risueño
Keyword(s):  
Liquid Medium ◽  
Embryo Development ◽  
Secretory Pathway ◽  
Microspore Culture ◽  
Microspore Embryogenesis ◽  
In Situ Monitoring ◽  
Subcellular Localisation ◽  
Embryo Cells

In vitro plant cells in culture release proteins and carbohydrates, but the active molecules responsible for sustaining the switch in embryogenic development and progression have not yet been identified. In maize (Zea mays L.), the Esr genes encode for small hydrophilic proteins and are expressed in the restricted region of maize endosperm surrounding the embryo: the embryo surrounding region (ESR). In the present work, the possible influence of secreted molecules in the liquid medium during microspore-derived embryo development and specifically, the presence of Esr proteins, has been analysed in maize microspore cultures. The study has been conducted with in situ monitoring of the structural and cellular organisation of developing embryos and the subcellular localisation of the Esr proteins by immunofluorescence and immunogold labelling. The results obtained using confocal and electron microscopy revealed that Esr proteins were localised in elements of the secretory pathway and cell walls in microspore-derived embryo cells during early embryogenesis. Esr proteins were also detected in the liquid medium of maize microspore cultures and accumulated at 20 days of culture. Tunicamycin treatment to block protein glycosilation and, therefore, secretion inhibited microspore-derived embryo development, which was subsequently recovered by supplementation with medium containing all the secreted factors from a well developed microspore culture. Esr labelling was not present in non-developing microspore embryos of cultures treated with tunicamycin, whereas labelling was present again in the Golgi elements and secretory vesicles of embryo cells when development was restored. The results indicate that Esr proteins are part of the secreted proteins, which show a nursing or signalling role during in vitro embryo development in maize microspore embryogenesis cultures and provide new evidence for an endosperm-like function of microspore-derived embryo structures during the early stages.

scholarly journals Embryogenesis of European Radish (Raphanus sativus L. subsp. sativus convar. radicula) in Culture of Isolated Microspores In Vitro

Plants ◽  
2021 ◽  
Vol 10 (10) ◽  
pp. 2117
Author(s):  
Elena Victorovna Kozar ◽  
Elena Alekseevna Domblides ◽  
Alexsey Vasilevich Soldatenko
Keyword(s):  
Microspore Culture ◽  
Entire Family ◽  
Stage Of Development ◽  
Globular Stage ◽  
Isolated Microspores ◽  
Raphanus Sativus L ◽  
In Vitro Microspore Culture ◽  
First Time ◽  
Brassicaceae Family

The European radish is one of the most unresponsive crops in the Brassicaceae family to embryogenesis in in vitro microspore culture. The aim of this work was to study the process of embryogenesis of European radish and its biological features. In this study, the embryogenesis of European radish is described in detail with illustrative data for the first time. For the first time for the entire family Brassicaceae, the following were found: microspores with intact exines with ordered-like divisions; microspores completely free of exines; and a new scheme of suspensors attachment to the apical parts of embryoids. The morphology of double and triple twin embryoids was described, and new patterns of their attachment to each other were discovered. Uneven maturation of European radish embryoids at all stages of embryogenesis was noted. The period of embryoid maturation to the globular stage of development corresponded, in terms of time, to the culture of B. napus, and into the cotyledonary stage of development, maturation was faster and amounted to 17–23 days. The rate of embryoid development with and without suspensors was the same.

scholarly journals Cytological methods of analysis of haploid plants-regenerants of cabbage (Brassica oleracea L.) obtained in vitro

2019 ◽  
pp. 13-15
Author(s):  
Rima N. Kirakosyan ◽  
Elena A. Kalashnikovа
Keyword(s):  
Brassica Oleracea ◽  
Doubled Haploids ◽  
Reproductive Organs ◽  
Universal Method ◽  
Mineral Salts ◽  
Methods Of Analysis ◽  
White Cabbage ◽  
Regenerated Plants ◽  
Haploid Plants

Relevance Currently, in genetic studies and selection of cabbage cultures, biotechnological methods for creating clean lines — doubled haploids in the culture of anthers and in the culture of isolated microspores are widely used. A common feature of these technologies is that the plants obtained in vitro have different levels of ploidy and along with doubled haploids there are haploid, tetraploid and mixoploid forms. Therefore, the use of new cytological methods of analysis of haploid plants remains an urgent problem. Material and method The aim of this work is to establish the genetic nature of regenerated plants of Brassica oleracea L., obtained from reproductive organs in vitro. Isolated anthers and ovaries of white cabbage were cultivated on solid nutrient media containing mineral salts according to the recipe Murashige and Skoog (MS). The obtained regenerated plants were used to calculate the number of chromosomes in the root meristem, as well as the number of chloroplasts in the cells of the closing stomata of leaves using the new universal method of preparing preparations of plant chromosomes – “SteamDrop”. Results As a result of the research, the dependence of the level of ploidy on the cultivation conditions was studied. It has been shown that plants-regenerants of white cabbage, obtained in vitro from reproductive organs, had a different set of chromosomes (n, 3n, 5n). It was established that the number of chloroplasts in the stomatal cells of regenerated plants was from 9 to 45, while the original donor plants had 18–20.

scholarly journals Microspore embryogenesis in vitro: the role of stresses

2019 ◽  
Vol 23 (1) ◽  
pp. 86-94 ◽  
Author(s):  
T. I. Djatchouk ◽  
O. V. Khomyakova ◽  
V. N. Akinina ◽  
I. A. Kibkalo ◽  
A. V. Pominov
Keyword(s):  
Microspore Culture ◽  
Doubled Haploids ◽  
Embryo Sac ◽  
Microspore Embryogenesis ◽  
Gametic Embryogenesis ◽  
Chemical Pretreatments ◽  
Dh Lines

Gametic embryogenesis is one form of totipotency of plant cells, in which either male or female gametes are induced to form embryoids (sporophytes). Regeneration of haploid plants from embryoids and subsequent chromosome duplication result in doubled haploids and DH-lines. The production of haploids and doubled haploids (DHs) through gametic embryogenesis allows a single-stage development of complete homozygous lines from heterozygous plants. The development of effective haploid protocols to produce homozygous plants has a significant impact on plant breeding, shorting the time and costs required to establish new cultivars. There are several available methods to obtain haploids and DHs-lines, of which anther or isolated microspore culture in vitro are the most effective. Microspore embryogenesis is more commonly applied. This is in part because more male gametophytes are contained in a single anther compared to the single female gametophyte per embryo sac. Microspore embryogenesis is regarded as one of the most striking examples of plant cell totipotency. The switch of cultured microspores from gametophytic to sporophytic mode of development has been induced by stress treatments of various kinds applied to donor plants, inflorescences, buds, anthers or isolated microspores both in vivo and in  vitro. Physical or chemical pretreatments (cold and heat shock, sugar starvation, colchicine, n-butanol, gametocydes) act as a trigger for inducing the sporophytic pathway, preventing the gametophytic pathway development of microspore. The recent investigations have revealed that cold pretreatment during microspore reprogramming acts rather as an anti-stress factor alleviating the real stress caused by nutrient starvation of anthers or microspores isolated from donor plants. Under stress pretreatment a vacuolated and polarized microspore transformed into a depolarized and dedifferentiated cell, which is an obligatory condition for reprogramming their development. We summarize data concerning the role of various stresses in the induction of microspore embryogenesis and possible mechanisms of their action at cellular and molecular levels. Identification of new stresses allows creating efficient protocols of doubled haploid production for end-user application in the breeding of many important crops.

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