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Wheats --- haploidy --- Anther culture
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Triticum aestivum --- Anther culture --- Embryon androgenique
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Triticum aestivum --- Embryo culture --- Anther culture --- Morphogenesis --- Regeneration. --- Regeneration
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Androgenesis --- culture media --- In vitro culture --- Anther culture --- Lagenaria siceraria
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Grain legumes, particularly those belonging to the genus Phaseolus, are considered to be recalcitrant to tissue culture. The objective of this thesis was to develop a protocol of anther in vitro culture of several Phaseolus species, by considering in priority factors which improve androgenesis induction. The effect of the following parameters was studied : genetic origin of the donor plants, conditions of culture, development stage of the microspores, composition of the culture media, concentration and density of the anthers in the medium and cold pre-treatment of the flower buds or of the anthers. The available protocols can be applied either to cultivated or to wild genotypes of Phaseolus vulgaris and Phaseolus coccineus and the donor plants must be cultivated in controlled conditions. The best results were obtained with anthers containing microspores at the uninucleated stage. The anther response was influenced by the composition of the medium and was the highest with media containing the salts of Murashige and Skoog, kinetin (2 mg. l-1), 2, 4-D (2 mg. l-1), a sugar (either sucrose or maltose at a concentration ranging from 1.25 to 2.5 %), the vitamins of the N6 medium, casein hydrolysate and agarose. The best conditions were obtained by growing 40 anthers in Petri dishes with a diameter of 55 mm containing 10 ml of medium, in the dark and at 26°C, with a cold pre-treatment of the flower buds for 1 or 2 days. Preliminary trials enabled the identification of factors which could improve plant regeneration : the choice of genotype and composition of the medium. Globular structures were observed on calli developed on a medium containing Murashige and Skoog's salts, abscissic acid (1-2 mg. l-1), sucrose (3 %), vitamins of the N6 medium, glutamine (100 mg. l-1), casein hydrolysate (2 g. l-1) and agarose (5 g. l-1). Finally, the ploidy level of the calli was determined by n-microfluorometry and showed a high frequency of haploid cells.
Phaseolus vulgaris --- Phaseolus coccineus --- Anther culture --- In vitro culture --- culture media --- Variety trials --- breeding methods
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Gossypium --- Organ culture --- Hybridization --- Interspecific sterility --- haploidy --- Anther culture --- Clonal variation --- genetic engineering --- Secondary metabolites --- Tannins --- Plant biotechnology
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Wheats --- In vitro culture --- Anther culture --- Selection. --- Selection --- breeding methods --- Resistance to injurious factors --- Sodium chloride --- Salt tolerance --- Salinity --- Triticum --- Induced mutation
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Biotechnologie --- Biotechnology --- Horticulture --- horticulture --- Génétique --- genetics --- Ressource génétique --- genetic resources --- Transfert de gène --- Gene transfer --- Stress --- genomes --- Culture d'anthère --- Anther culture --- Transformation génétique --- genetic transformation --- California --- Plant biotechnology --- Congresses. --- Congresses --- Plant biotechnology - Congresses. --- Horticulture - Congresses. --- horticulture.
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Doubled haploids (DHs) are powerful tools to reduce the time and costs needed to produce pure lines to be used in breeding programs. DHs are also useful for genetic mapping of complex qualitative traits, to avoid transgenic hemizygotes, for studies of linkage and estimation of recombination fractions, for screening of recessive mutants. These are just some of the advantages that make DH technology one of the most exciting fields of present and future plant biotechnology. All of the DH methods have model species where these technologies have been developed, or that respond every efficiently to their corresponding induction treatment. However, not all the species of economical/agronomical interest respond to these methodologies as they should be in order to obtain DHs on a routine basis. Indeed, many of them are still considered as low-responding or recalcitrant to these treatments, including many of the most important crops worldwide. Although many groups are making significant progresses in the understanding of these intriguing experimental pathways, little is known about the origin, causes and ways to overcome recalcitrancy. It would be very important to shed light on the particularities of recalcitrant species and the special conditions they need to be induced. In parallel, the knowledge gained from the study of basic aspects in model species could also be beneficial to overcome recalcitrancy. In this e-book, we present a compilation of different approaches leading to the generation of DHs in model and in recalcitrant species, and different studies on new and relevant aspects of this process, useful to extract common traits and features, to know better these processes, and eventually, to elucidate how to make DH technology more efficient.
Plant science. --- Botanical science --- Phytobiology --- Phytography --- Phytology --- Plant biology --- Plant science --- Biology --- Natural history --- Plants --- Floristic botany --- anther culture --- Embryogenesis --- in vitro culture --- Microspore --- Pollen --- gametic --- androgenesis --- haploid --- microspore culture --- Doubled haploid
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La production de plantes haploïdes doublées (HD) permet aux sélectionneurs végétaux d’obtenir des lignées complètement homozygotes en une seule génération. Le laboratoire de culture in vitro de l’Institut de Genech est spécialisé dans la production de plantes HD chez plusieurs espèces. Chez le blé tendre (Triticum aestivum L.), ce laboratoire utilise en routine le croisement intergénérique avec le maïs pour produire des HD. Toutefois l’entreprise souhaiterait se pencher vers une méthode utilisant l’androgenèse : la culture d’anthères. En effet, cette technique permettrait de ne plus devoir gérer une culture de maïs et un doublement chromosomique spontané est possible via cette méthode. Dans ce contexte, l’objectif de cette étude est d’explorer différentes pistes afin de mettre en place un protocole de culture d’anthères de blé tendre. Quatre expérimentations ont été effectuées sur des plantes mères de serre. Une première expérience a comparé deux types de prétraitement : un choc à 4°C pendant deux semaines appliqué sur l’épi et un choc à 1.0M mannitol pendant quatre jours à 20°C sur les anthères. Les deux méthodes ont obtenu une réponse androgénique similaire. Ensuite, les trois autres expérimentations ciblaient la composition du milieu d’induction. Une comparaison entre un milieu semi-liquide, W14mf, et un milieu solide, C17 a tout d’abord été réalisée. Le milieu W14mf a produit en moyenne une plus grande quantité de structures embryonnaires (SE) et de plantes vertes (PV ; 37,32 SE/100 anthères (A) et 7,64 PV/100A) par rapport au milieu C17 (8,72 SE/100A et 2,24 PV/100A). De plus, l’utilisation du milieu W14mf pré-conditionné avec cinq ovaires a permis de tripler la production en SE alors que, de façon étonnante, aucune progression du nombre de PV n’a été notée. Enfin, un ajout de 25mg/L d’arabinogalactane non purifiée dans le milieu W14mf n’a pas eu d’impact sur la réponse androgénique des microspores. L’albinisme a constitué un obstacle majeur pour certaines expérimentations. Dans certains cas, plus de 95% des plantes régénérées étaient albinos. Le génotype des plantes mères est le principal facteur expliquant ce phénomène. Mais de nombreux facteurs externes, tels que les conditions physiologiques des plantes mères, ont également un impact sur l’albinisme. Dès lors, une expérimentation à petite échelle a été effectuée pour comparer la réponse androgénique d’anthères provenant de plantes mères du champ et mis en culture sans prétraitement avec celle d’anthères provenant de plante mères de serre et mis en culture après un prétraitement des épis durant 2 semaines à 4°C. Les plantes mères de champs ont produit significativement plus de PV (10,64 PV/100A) par rapport aux plantes mères de serre (4,22 PV/100A). En conclusion, l’ensemble de ces expériences nous a permis d’affirmer que l’avenir de la culture d’anthères pour la production d’HD est prometteur au sein du laboratoire de l’Institut de Genech. Néanmoins, il sera nécessaire de trouver des solutions pour restreindre au maximum les deux principales limites de la culture d’anthères : l’albinisme et la dépendance au génotype des plantes mères. The production of doubled haploid (DH) plants enables plant breeders to obtain completely homozygous lines in a single generation. The in vitro culture laboratory of Genech Institute is specialized in the production of DH plants on several species. In common wheat (Triticum aestivum L.), this laboratory routinely uses the intergeneric cross with maize to produce DH. However, the company would like to look into a method which uses androgenesis: the anther culture. Indeed, this technique would eliminate the need to manage a maize culture and a spontaneous chromosomal doubling is possible via this method. In this context, the aim of this study was to explore different clues in order to implement an anther culture protocol on common wheat. Four experimentations have been carried out on mother plants coming from the greenhouse. A first experiment compared two types of pretreatment: a 4°C shock applied during two weeks on spike and a 1.0M mannitol shock during four days at 20°C on anthers. The two methods displayed a similar androgenic response. Then, the three other experimentations were focused on the composition of the induction medium. A comparison between a semi-liquid medium, W14mf, and a solid medium, C17, was firstly done. The W14mf medium produced in average more embryo-like structures (ELS) and green plants (GP; 37.32 ELS/100 anthers (A) and 7.64 GP/100A) than C17 medium (8.72 ELS/100A and 2.24 GP/100A). Moreover, the use of pre-conditioned W14mf medium with five ovaries helped to triple the production of ELS, whereas surprisingly, no increase in the number of GP was recorded. Lastly, an addition of 25 mg/L of non-purified arabinogalactan in the W14mf medium didn’t have an impact on the androgenic response of microspores. The albinism formed a major obstacle for some experiments. In some cases, more than 95% of the regenerated plants were albinos. The main factor explaining this phenomenon is the genotype of mother plants. But a lot of external factors, such as the physiological conditions of mother plants, have also an impact on albinism. Therefore, a small-scale experiment was undertaken to compare the androgenic response of anthers of mother plants originating from the field and in which no stress pretreatment was done, with mother plants coming from the greenhouse and in which a 4°C pretreatment was done on spikes during two weeks. The mother plants of the field produced significantly more GP (10.64 GP/100A) than mother plants of the greenhouse (4.22 PV/100). To conclude, this set of experimentations allowed us to reveal that the future of anther culture to generate DH seems promising within the laboratory of Genech Institute. Nevertheless, it will be necessary to find solutions to limit as much as possible the two main constraints of anther culture: the albinism and the dependence on mother plants genotype.
Doubled haploid --- Androgenesis --- Anther culture --- Triticum aestivum L. --- Common wheat --- Haploïde doublé --- Androgenèse --- Culture d'anthères --- Triticum aestivum L. --- Blé tendre --- Sciences du vivant > Biologie végétale (sciences végétales, sylviculture, mycologie...) --- Sciences du vivant > Agriculture & agronomie
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