Embryoid Production Research Articles

Isolated microspore culture for embryoid production in Artemisia annua L.

Isolated microspore culture for embryoid production in Artemisia annua L.

Carrot (Daucus carota L.) In Vitro Regeneration

Carrot (Daucus carota L.) In Vitro Regeneration Experiments were carried out for optimization of conditions for morphogenic callus production and regeneration of carrot (Daucus carota L.) using four cultivars cultivated in Iran. Hypocotyl segments were put on media supplemented with 0.2 mg·L-1 2,4-D for callus initiation and proliferation. Small portions of callus (25 mg) were cut and transferred to new media with different concentrations of 2,4-D including 0.2, 0.5 and 1 mg·L-1. MS (Murashige & Skoog 1962) and MSm (Masuda et al. 1981) media were used in the experiments to identify the differences between these two media according to callus, embryoid and plantlet production. Obtained results showed that low level of 2,4-D (0.2 mg·L-1) was more effective in morphogenic callus production although higher amounts of it (0.5 mg·L-1 and 1 mg·L-1) caused more callus growth. Kinetin in concentration of 0.1 mg·L-1 was more effective for regeneration than BAP used in concentration 1 mg·L-1 especially according to embryoid production. MSm medium was more useful than MS for callus production and regeneration of carrot plantlets. Among tested cultivars Nantes Improved had more capability to produce viable plantlets.

Improvement of isolated microspore culture of pepper (Capsicum annuum L.) via co-culture with ovary tissues of pepper or wheat

The influence of the developmental stage of microspores on establishing isolated microspore cultures of three Hungarian (‘Szegedi 80’, ‘Szegedi 178’, and ‘Remeny’) and three Spanish (‘Jeromin’, ‘Jariza’, and ‘Jaranda’) pepper genotypes was investigated. Donor anthers containing 80% uninucleated and 20% binucleated microspores yielded the highest frequency of successful microspore cultures. Co-cultures with wheat, line ‘CY-45’, ovaries exhibited enhanced frequency of embryoid production than those with pepper ovaries. Differences in efficiency of isolated pepper microspore culture establishment were observed among different pepper genotypes. Green plantlets were regenerated from microspore-derived embryoids, but some were exhibited abnormal growth habits, such as leaf rosetting. A total of seven fertile microspore-derived plants were obtained, including three ‘Jariza’, three ‘Jaranda’, and a single ‘Szegedi 80’ plant.

Improvement of Citrus clementina Hort. ex Tan. microspore-derived embryoid induction and regeneration.

An improvement of the protocol for haploid induction through anther culture of Citrus clementina Hort. ex Tan. cv. Nules was achieved following the evaluation of a number of the factors affecting androgenesis. The influence of thidiazuron (TDZ) and three temperature pre-treatments (4 degrees C, 25 degrees C, 32 degrees C) on the floral buds with respect to anther culture of C. clementina Hort. ex Tan., cv. Nules was investigated. An increased embryoid production was induced in the medium supplemented with TDZ. Pre-treatment temperatures of 4 degrees C and 25 degrees C were more favorable for embryo production than 32 degrees C. Regeneration of androgenic haploid plantlets from cv. SRA 63 of C. clementina is reported here for the first time.

Microspore culture in wheat (Triticum aestivum) – doubled haploid production via induced embryogenesis

The inherent potential to produce plants from microspores or immature pollen exists naturally in many plant species. Some genotypes in hexaploid wheat (Triticum aestivum L.) also exhibit the trait for androgenesis. Under most circumstances, however, an artificial manipulation, in the form of physical, physiological and/or chemical treatment, need to be employed to switch microspores from gametophytic development to a sporophytic pathway. Induced embryogenic microspores, characterized by unique morphological features, undergo organized cell divisions and differentiation that lead to a direct formation of embryoids. Embryoids `germinate' to give rise to haploid or doubled haploid plants. The switch from terminal differentiation of pollen grain formation to sporophytic development of embryoid production involves a treatment that halts gametogenesis and initiates sporogenesis showing predictable cellular and molecular events. In principle, the inductive treatments may act to release microspores from cell cycle control that ensures mature pollen formation hence overcome a developmental block to embryogenesis. Isolated microspore culture, genetic analyses, and studies of cellular and molecular mechanisms related to microspore embryogenesis have yielded useful information for both understanding androgenesis and improving the efficiency of doubled haploid production. The precise mechanisms for microspore embryogenesis, however, must await more research.

Regeneration of fertile doubled haploid plants from colchicine‐supplemented media in wheat anther culture

AbstractThe effect of colchicine added to induction medium for the production of fertile doubled haploid plants after in‐vitro anther culture was studied in wheat, Triticum aestivum L. For this, one winter and two spring wheat varieties were used. Anther cultures of the three genotypes were treated with 0.03% colchicine for 3 days at the beginning of microspore induction. Colchicine had no significant effect on anther response and embryoid production of the genotypes examined. However, in the winter wheat genotype ‘Mv Szigma’, colchicine caused a significant reduction in microspore‐derived structures. A significant decrease was also observed in plant regeneration ability of two genotypes (‘Vergina’ and ‘Acheloos’) after colchicine treatment. In addition, a significant reduction of the albinos produced was observed in all genotypes after olchicine treatment. In contrast, the regenerants obtained from the colchicine‐supplemented induction media produced significantly higher percentages of fertile plants in all genotypes. However, the level of fertility, was significantly different among the fertile plants obtained. This, together with the observation that in the case of the winter wheat variety the colchicine treatment resulted in 100% completely fertile plants with a high seed‐setting ability indicate that there is space for further improvement of the method when it is applied to spring cultivars. Finally, the increased number of seeds per 100 plated anthers obtained from all three genotypes after colchicine treatment, clearly demonstrates that the addition of colchicine to induction medium was superior to the conventional anther culture method and it could therefore be introduced into wheat breeding programmes.

Analysis of anther culture response in hexaploid triticale

AbstractThe androgenic response of five varieties and 10 F1 intervarietal hybrids of 6 × triticale was analysed. The overall response involved three independent phenomena: the production of embryoids, the capability of embryoids to regenerate plants, and the production of green plants. Each of these factors appeared to be controlled by independent genetic systems. From a practical point of view, the product of these three factors, the number of green haploid plants per anther (GPA), is the most reliable variable for the evaluation of genotypes. Positive heterotic effects on GPA were observed in the hybrids, which may indicate that GPA is controlled predominantly by additive genes. No correlation was found between the number of green haploid plants produced by hybrids and that yielded by the best parental line. Therefore, it is difficult to predict the yield of green haploid plants that can be produced by a hybrid simply from the number produced by its parents.

The Effect of Calcium on Ethylene Production and M icrospore-derived Embryogenesis in Barley ( Hordeum vulgare L.) and Wheat ( Triticum aestivum L.) Anther Cultures

The roles of calcium on microspore-derived embryo (embryoid) formation and ethylene production in barley ( Hordeum vulgare L. cv. Bruce) and wheat ( Triticum aestivum L. cv. Chris) anther cultures were investigated. The inclusion of calcium as CaCl 2 in the medium increased embryoid production, at an optimal concentration of 1 mM in barley. The addition of the ionophore A23187 (0.5 μM) with CaCl 2 (1 mM) further improved the embryoid formation in both wheat and barley. Since exogenous calcium and ionophore increased ethylene production measured at culture initiation time, it was suggested that the positive action of free calcium on embryoid formation might be achieved through initial ethylene production and stimulation of tissue senescence.

Genetic analysis for haploid‐regenerations responses of the hexaploid‐wheat anther cultures

AbstractGenetic variability in response to anther culture was investigated in 49 winter hexaploid wheats, comprising 33 pure lines (F10) derived from a composite cross programme and their 16 parental genotypes. All genotypes were grown in a randomized block design with three replications in a controlled greenhouse. The number of embryoids and total plant regeneration per 100 anthers, as well as the numbers of green and albino plants regenerated per 100 embryoids, were measured.Significant genetic variability was observed among the 49 genotypes for all the traits studied. All traits showed high heritability. Among the genotypes compared, DC230N and 1BPT‐40 gave the best results for the production of embryoids and IBPT‐78 had the highest value for the production of green plants.The genotype IBPT‐34 developed a large number of albino plants, and it should be useful as a parent in studies to determine the genetic control of albino plants in wheat.

Characterisation of protoplasts from hypocotyls of Helianthus annuus in relation to their tissue origin

Cells and protoplasts isolated from three different tissues of sunflower hypocotyls and cultured either in liquid or agarose medium were compared in terms of their volume, DNA content, division potential and embryoid formation. Epidermal and external cortical cells differ from other tissue cells by their small size, their weak response to plasmolysis and their low DNA content (around 1C). They contribute only very weakly to the dividing protoplast population. In contrast, protoplasts from cortical and medullar cells both have similar division potential, reaching 50%. The nuclear DNA content of these two cell types, as well as their corresponding protoplasts, has a 2C value, taking root tip cells in G0 phase as standard. The culture conditions induce the same specific response in protoplasts isolated from both tissues: exclusively loose colony formation in liquid medium, and mainly production of embryoids in agarose medium.

Chloroplast Genome Breakdown in Microspore Cultures of Barley ( Hordeum vulgare L.) Occurs Primarily during Regeneration

Southern blot analyses of DNA from microspore cultures of the barley variety Alexis suggested that plastid genome deletions/rearrangements causing albinism occur primarily during regeneration. The Alexis variety responded to microspore culture largely by production of ill-defined embryoids and callus from which plants could be regenerated with a low frequency. Only 0.06 green and 0.61 albino plants were regenerated per anther in contrast to the Igri variety, which formed well-defined embryoids that readily regenerated 11.7 green and 0.1 albino plants per anther. In Alexis, the plastid genomes appeared to remain intact during the microspore culture but started to break down in the structures that underwent regeneration. This development accelerated in parallel with differentiation and leaf formation. In Igri microspore cultures, callus and albino plant formation could be induced by elevated temperatures and in particular during the first 10 days of culture and during the regeneration phase.

Protein markers for anther culturability in barley

Two-dimensional electrophoresis of proteins from a recombinant population of anther culture-derived doubled haploid lines identified 4 loci or linkage groups showing a deviation from an expected 1∶1 segregation. It was hypothesized that these markers are linked to genes involved in the process of haploid plant production and that the deviation was due to a selection for alleles conferring higher anther culture response. To check this hypothesis, the anther culturability of 50 of the doubled haploid lines and their two inbred parents was assessed. It was found that 2 of the loci which had a distortion of segregation showed a significant effect on anther culture response, the most efficient allele being the most frequent in both loci. In addition, 2 more markers associated with anther culturability were found. One of the first mentioned 2 loci and one of the latter 2 were found to be linked to genes involved in both embryoid production and subsequent green plant regeneration. The remaining two were linked to genes involved only in green plant regeneration. Of the 4 favorable alleles 3 were inherited from one parent.

Increase of callus and embryoid production from hypocotyl protoplasts of sunflower (Helianthus annuus L.) by culture in microdrops

Hypocotyls of seedling plants of the sunflower inbred line HNK-81 were used as a source of protoplasts. Optimal conditions of isolation and culture of protoplasts were established. Using the method of individual culture in microdrops, a 77% final plating efficiency was achieved. Cytokinins, especially zeatin, showed a significant influence on the formation of globular embryo-like structures, but these failed to develop into mature embryos. Calli obtained in liquid media containing auxins and cytokinins grew on agar solidified media without growth regulators.

The effects of silver nitrate, colchicine, cupric sulfate and genotype on the production of embryoids from anthers of tetraploid wheat (Triticum turgidum)

Anther culture of four tetraploid wheat (Triticum turgidum) genotypes was studied using ten different culture medium treatments in a randomized block design with four replicates. Each replicate consisted of 2 pots with 3 plants. Anther donor plants were grown in a greenhouse with a 16 h day/8 h night at 25°C and 15°C, respectively. The first treatment which was considered as the control, was potato 2 medium modified by adding 0.5 g l−1 glutamine and solidified by gelrite (4 g l−1). The nine test treatments differed from the control by addition of 3 different concentrations of silver nitrate (1, 2.5 and 5 mg l−1), colchicine (10, 100 and 200 mg l−1) or cupric sulfate (2,5 and 10 mg l−1). The study of about 2000 anthers per genotype and treatment showed that both genotype and treatment affected embryoid formation. The presence of cupric sulfate (10 mg l−1) and silver nitrate (2.5 and 5 mg l−1) usually increased the frequency of embryoid formation in 3 genotypes out of the 4 studied. On the contrary, colchicine had a significant and enegative effect on anther culture responses for three out of the four genotypes studied. Because of the large genotype x medium interaction, it is very difficult to identify the best medium for embryo production by all genotypes studied.

Isolation and characterization of potato diploid clones generating a high frequency of monohaploid or homozygous diploid androgenetic plants

Cultured microspores of diploid potato clones lead with high frequency to diploid regenerants. In this paper we report on the genetic variability for in-vitro monohaploid production from anthers of diploid plants. Three diploid genotypes have been isolated which combine the capacity to regenerate monohaploid plants with outstanding embryoid production. A trait of the anther-donor clones associated with the generation of monohaploid plants is the low production of 2n pollen grains. When present in anthers of diploid genotypes, diploid unreduced microspores are, in fact, derived mainly from a first division restitution mechanism leading to high heterozygosity of the derived embryoids, a state which apparently supports superior growth in-vitro. Also, reduced microspores have been found capable of generating diploid regenerants and the adoption of the RFLP technique allowed the isolation of such diploid plants, which can be considered to be pure lines. Donor clones with a low capacity to generate monohaploids are, as expected, poor producers of homozygous diploid plants. The selection of an anther donor producing a sufficient number of monohaploid or homozygous diploid regenerants fulfills the requirements of the first part of the analytical breeding scheme, i.e., the production of homozygous diploid clones.

The Effect of Sugars and Growth Regulators on Embryoid Formation and Plant Regeneration from Barley Anther Culture

AbstractThe influence of carbon source and growth regulator composition in induction medium on anther culture response was investigated using spring barley genotypes. Anthers were cultured on BAC3, Ficoll‐containing medium, supplemented with one of the following carbohydrates: sucrose, maltose, cellobiosc and melibiose (6 % w/v). The use of either maltose or cellobiose resulted in a significantly higher anther response, calli and/or embryoid production and green plant regeneration compared to the incubation of anthers on a medium containing sucrose. Contrary to these results, the replacement of sucrose by melibiose in BAO medium, drastically reduced the efficiency of anther culture. As an average for the three genotypes tested, the frequency of green plants per 100 anthers plated was 9‐ to 22‐fold higher on medium supplemented with sucrose or cellobiose than on medium containing melibiose as a sole carbohydrate.Among the growth regulators tested, the combination of auxin NAA (2 mg/l) and cytokinin BAP (1 mg/1) performed much better than the employment of auxin 2,4‐D combined either with zeatin riboside or BAP as cytokinins. The beneficial effect of medium supplemented with NAA and BAP was associated with better embryoid formation compared to the other growth regulator combinations tested. The hormone‐free combination gave a similar anther response and production of calli as any medium supplemented with growth regulators, but the regeneration capacity of calli produced on hormone‐free medium was much lower, resulting in the drastic reduction of the number of both total and green regenerants.

The influence of culture conditions on anther culture response of commercial varieties of Solanum tuberosum L.

The effect of media manipulatioss, temperature pretreatment, carbohydrate source, and seasonal variation on tetraploid potato anther cultures was investigated. The anther culture responses of three commercial Nordic potato varieties from Scandinavia and two from Germany were compared on different media manipulations. With most of the varieties, solid MS media gave better yields than other published media manipulations. Pretreatments at +6°C and at +30°C were studied on Pito and Danva varieties. The +6°C pretreatment and no pretreatment had the same effect on the anther culture response of cv. Pito, while with cv. Danva pretreatment at +6°C promoted embryogenesis. The +30°C pretreatment had no positive effect on anther culture response on either cultivar. The effect of maltose, melibiose and mannitol individually and in combination with sucrose were compared to normal sucrose medium in cv. Pito anther cultures. Anthers incubated on normal sucrose medium gave the highest embryoid and plant yields; the second highest plant yields were obtained on pure maltose medium. Strong seasonal variation was observed throughout the year in cv. Pito anther cultures. The percentage of anthers producing embryoids ranged from 15–20% during September and October to just 1–3% from February through May. The annual average embryoid production rate was 6.18%.

Relationship of Senescence to Androgenesis in Barley ( Hordeum vulgare L. cv. Klages)

Summary During anther culture, lipid peroxidation was used as measure of senescence to determine if there was any correlation with androgenesis. The production of thiobarbituric acid reactive substances (TBA-RS) and the saturation of total fatty acids were observed. The inclusion of free radical scavengers (hydroquinone, vitamin E) in the medium not only delayed and suppressed lipid peroxidation and ethylene production during the early culture period but also reduced embryoid production. It is concluded that early lipid peroxidation, as a general symptom of senescence at the time of culture initiation, was beneficial to androgenesis in barley relative to the culture medium and genotype used.

Effects of Growth Regulators and Genotypes on Callus and Embryoid Induction from Maize Anther Culture *

Fifty genotypes and ten growth-regulator combinations were used in two experiments (I and II) to investigate genotype and hormonal effects on production of callus and embryoids via anther culture in maize (Zea mays L.). Hormonal effects across all genotypes were not significant in either experiment. However, highest callus-induction frequencies in both experiments occurred on YP basal medium plus 2,4-D at 2.0 mg 1−1, and kinetin at 1.5 mg 1−1 indicating that this combination was more effective than others. Genotypic differences in callus or embryoid induction across all media were significant. The most responsive genotypes in experiment I were single-cross hybrids Yuanwu × 592 and K727 × K305, which produced 18.3 and 6.7 % calli, respectively, with their appropriate media. The most responsive entry in experiment II was CIMMYT Pool 29, which produced 15.0 % calli on appropriate medium and an average of 10.0 % calli across 10 media. Twenty-three plantlets was regenerated from this study. Most of them developed embryogenically.

Rapid‐cycling Brassica species: anther culture potential of B. campestris L. and B. napus L.

summaryA study was made of the production of embryoids by cultured anthers of rapid‐cycling populations of Brassica campestris L. and Brassica napus L. In the case of B. campestris, best results were obtained when anther donors were grown at 24 °C with continuous light; anthers were excised from 1.6–2.00 mm buds with the pollen at the mid‐uninucleate stage of development and were cultured on a solidifed Brassica anther culture medium of Keller & Armstrong (K), and when incubation was at 35 °C for 1 day, followed by 30 °C for 1 day and then continuous incubation at 25 °C. The response to anther culture of B. napus was generally very poor compared with that of B. campestris but the best results were obtained when anther donors were grown at 18 °C with continuous light; anthers were excised from 2.1–2.5 mm long buds with the pollen at the mid‐uninucleate stage of development, were cultured on a liquid formulation of K medium, and incubation was at 35 °C for 1 day, followed by 30 °C for 6 days and then continuous incubation at 25 °C.