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1.
Methods Mol Biol ; 2289: 263-270, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34270076

RESUMO

The production of doubled haploid (DH) plants from microspores is an important technique used in plant breeding and basic research. DH technology is a rapid method for developing homozygous lines, which can be used to accelerate crop improvement programs. Haploidy technology can also be used in mutagenesis, transformation, and basic research such as genomic, biochemical, and physiological studies. There is no general protocol that will result in the production of DH in all species, as differences occur among species and among genotypes within a species in terms of embryogenic response. Here we describe methodology for developing doubled haploids in cow cockle (Saponaria vaccaria L.).


Assuntos
Melhoramento Vegetal/métodos , Saponaria/genética , Vaccaria/genética , Flores/genética , Genótipo , Haploidia , Mutagênese/genética
2.
Methods Mol Biol ; 2288: 103-111, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34270007

RESUMO

Doubled haploidy technology is a powerful tool to accelerate the breeding of new crop varieties. Protocols are not universal, as even species within the same family require a specific process. Here we describe methods for developing doubled haploids for fennel and dill, both Apiaceae species which are used for food, flavorings, and medicine.


Assuntos
Anethum graveolens/crescimento & desenvolvimento , Anethum graveolens/genética , Foeniculum/crescimento & desenvolvimento , Foeniculum/genética , Melhoramento Vegetal/métodos , Produtos Agrícolas/genética , Produtos Agrícolas/crescimento & desenvolvimento , Meios de Cultura/química , Diploide , Haploidia , Homozigoto , Biologia Molecular/métodos , Pólen/genética , Pólen/crescimento & desenvolvimento , Técnicas de Cultura de Tecidos
3.
Methods Mol Biol ; 2072: 183-198, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-31541447

RESUMO

CRISPR/Cas9-based genome editing technology has the potential to revolutionize agriculture, but many plant species and/or genotypes are recalcitrant to conventional transformation methods. Additionally, the long generation time of crop plants poses a significant obstacle to effective application of gene editing technology, as it takes a long time to produce modified homozygous genotypes. The haploid single-celled microspores are an attractive target for gene editing experiments, as they enable generation of homozygous doubled haploid mutants in one generation. Here, we describe optimized methods for genome editing of haploid wheat microspores and production of doubled haploid plants by microspore culture.


Assuntos
Sistemas CRISPR-Cas , Edição de Genes , Haploidia , Mutagênese , Triticum/genética , Regulação da Expressão Gênica no Desenvolvimento , Regulação da Expressão Gênica de Plantas , Genoma de Planta , Genótipo , Desenvolvimento Vegetal/genética , Plantas Geneticamente Modificadas , RNA Guia
4.
Phytochemistry ; 69(15): 2678-88, 2008 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-18823922

RESUMO

Developing seeds of Brassica napus contain significant levels of ABA and products of oxidation at the 7'- and 9'-methyl groups of ABA, 7'- and 9'-hydroxy ABA, as well stable products of oxidation of the 8'-methyl group, phaseic acid and dihydrophaseic acid. To probe the biological roles of the initially formed hydroxylated compounds, we have compared the effects of supplied ABA and the hydroxylated metabolites in regulating oil synthesis in microspore-derived embryos of B. napus, cv Hero that accumulate long chain fatty acids. Uptake into the embryos and metabolism of each of the hormone metabolites was studied by using deuterium labeled analogs. Supplied ABA, which was rapidly metabolized, induced expression of oleosin and fatty acid elongase genes and increased the accumulation of triacylglycerols and very long chain fatty acids. The metabolites 7'- and 9'-hydroxy ABA had similar effects, with the 9'-hydroxy ABA having even greater activity than ABA. The principal catabolite of ABA, 8'-hydroxy ABA, also had hormonal activity and led to increased oil synthesis but induced the genes weakly. These results indicate that all compounds tested could be involved in lipid synthesis in B. napus, and may have hormonal roles in other ABA-regulated processes.


Assuntos
Ácido Abscísico/metabolismo , Brassica napus/metabolismo , Hormônios/metabolismo , Óleos/metabolismo , Sementes/metabolismo , Esporos/metabolismo , Ácido Abscísico/farmacologia , Acetiltransferases/metabolismo , Brassica napus/embriologia , Brassica napus/genética , Elongases de Ácidos Graxos , Ácidos Graxos Monoinsaturados/metabolismo , Regulação da Expressão Gênica de Plantas/efeitos dos fármacos , Hormônios/farmacologia , Proteínas de Plantas/genética , Sementes/embriologia , Sementes/genética , Triglicerídeos/metabolismo
5.
J Exp Bot ; 59(10): 2857-73, 2008.
Artigo em Inglês | MEDLINE | ID: mdl-18552352

RESUMO

Brassica napus cultivar Westar is non-embryogenic under all standard protocols for induction of microspore embryogenesis; however, the rare embryos produced in Westar microspore cultures, induced with added brassinosteroids, were found to develop into heritably stable embryogenic lines after chromosome doubling. One of the Westar-derived doubled haploid (DH) lines, DH-2, produced up to 30% the number of embryos as the highly embryogenic B. napus line, Topas DH4079. Expression analysis of marker genes for embryogenesis in Westar and the derived DH-2 line, using real-time reverse transcription-PCR, revealed that the timely expression of embryogenesis-related genes such as LEAFY COTYLEDON1 (LEC1), LEC2, ABSCISIC ACID INSENSITIVE3, and BABY BOOM1, and an accompanying down-regulation of pollen-related transcripts, were associated with commitment to embryo development in Brassica microspores. Microarray comparisons of 7 d cultures of Westar and Westar DH-2, using a B. napus seed-focused cDNA array (10 642 unigenes), identified highly expressed genes related to protein synthesis, translation, and response to stimulus (Gene Ontology) in the embryogenic DH-2 microspore-derived cell cultures. In contrast, transcripts for pollen-expressed genes were predominant in the recalcitrant Westar microspores. Besides being embryogenic, DH-2 plants showed alterations in morphology and architecture as compared with Westar, for example epinastic leaves, non-abscised petals, pale flower colour, and longer lateral branches. Auxin, cytokinin, and abscisic acid (ABA) profiles in young leaves, mature leaves, and inflorescences of Westar and DH-2 revealed no significant differences that could account for the alterations in embryogenic potential or phenotype. Various mechanisms accounting for the increased capacity for embryogenesis in Westar-derived DH lines are considered.


Assuntos
Brassica napus/genética , Plantas Geneticamente Modificadas/genética , Pólen/genética , Brassica napus/citologia , Brassica napus/metabolismo , Cruzamento , Flores/citologia , Flores/genética , Flores/metabolismo , Regulação da Expressão Gênica de Plantas , Análise de Sequência com Séries de Oligonucleotídeos , Fenótipo , Reguladores de Crescimento de Plantas/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Plantas Geneticamente Modificadas/metabolismo , Pólen/citologia , Pólen/metabolismo , Transcrição Genética
6.
Plant Physiol ; 144(1): 134-54, 2007 May.
Artigo em Inglês | MEDLINE | ID: mdl-17384168

RESUMO

Isolated microspores of Brassica napus are developmentally programmed to form gametes; however, microspores can be reprogrammed through stress treatments to undergo appropriate divisions and form embryos. We are interested in the identification and isolation of factors and genes associated with the induction and establishment of embryogenesis in isolated microspores. Standard and normalized cDNA libraries, as well as subtractive cDNA libraries, were constructed from freshly isolated microspores (0 h) and microspores cultured for 3, 5, or 7 d under embryogenesis-inducing conditions. Library comparison tools were used to identify shifts in metabolism across this time course. Detailed expressed sequence tag analyses of 3 and 5 d cultures indicate that most sequences are related to pollen-specific genes. However, semiquantitative and real-time reverse transcription-polymerase chain reaction analyses at the initial stages of embryo induction also reveal expression of embryogenesis-related genes such as BABYBOOM1, LEAFY COTYLEDON1 (LEC1), and LEC2 as early as 2 to 3 d of microspore culture. Sequencing results suggest that embryogenesis is clearly established in a subset of the microspores by 7 d of culture and that this time point is optimal for isolation of embryo-specific expressed sequence tags such as ABSCISIC ACID INSENSITIVE3, ATS1, LEC1, LEC2, and FUSCA3. Following extensive polymerase chain reaction-based expression profiling, 16 genes were identified as unequivocal molecular markers for microspore embryogenesis in B. napus. These molecular marker genes also show expression during zygotic embryogenesis, underscoring the common developmental pathways that function in zygotic and gametic embryogenesis. The quantitative expression values of several of these molecular marker genes are shown to be predictive of embryogenic potential in B. napus cultivars (e.g. 'Topas' DH4079, 'Allons,' 'Westar,' 'Garrison').


Assuntos
Brassica napus/genética , Desenvolvimento Embrionário/genética , Genes de Plantas , RNA Mensageiro/metabolismo , Brassica napus/embriologia , Brassica napus/crescimento & desenvolvimento , DNA Complementar/metabolismo , Etiquetas de Sequências Expressas , Perfilação da Expressão Gênica , Marcadores Genéticos , Reação em Cadeia da Polimerase Via Transcriptase Reversa
7.
Plant Mol Biol ; 61(1-2): 269-81, 2006 May.
Artigo em Inglês | MEDLINE | ID: mdl-16786306

RESUMO

The Polima (pol) system of cytoplasmic male sterility (CMS) and its fertility restorer gene Rfp are used in hybrid rapeseed production in Brassica napus. To facilitate map-based cloning of the Rfp gene, we have successfully transferred the pol cytoplasm and Rfp from the amphidiploid B. napus to the diploid species B. rapa and generated a doubled haploid pol cytoplasm B. rapa population that segregates for the Rfp gene. This was achieved through interspecific crosses, in vitro rescue of hybrid embryos, backcrosses, and microspore culture. Male fertility conditioned by Rfp was shown to co-segregate in this population with Rfp-specific mitochondrial transcript modifications and with DNA markers previously shown to be linked to Rfp in B. napus. The selfed-progeny of one doubled haploid plant were confirmed to be characteristic B. rapa diploids by cytogenetic analysis. Clones recovered from a genomic library derived from this plant line using the RFLP probe cRF1 fell into several distinct physical contigs, one of which contained Rfp-linked polymorphic restriction fragments detected by this probe. This indicates that chromosomal DNA segments anchored in the Rfp region can be recovered from this library and that the library may therefore prove to be a useful resource for the eventual isolation of the Rfp gene.


Assuntos
Brassica napus/genética , Brassica rapa/genética , Clonagem Molecular , Genes de Plantas , Haploidia , Brassica napus/anatomia & histologia , Brassica napus/metabolismo , Brassica rapa/anatomia & histologia , Brassica rapa/metabolismo , Mapeamento Cromossômico , Cromossomos de Plantas/ultraestrutura , Cosmídeos/genética , Fertilidade/genética , Marcadores Genéticos , Biblioteca Genômica , Hibridização Genética , Meiose , Proteínas Mitocondriais/genética , Proteínas Mitocondriais/metabolismo , Polimorfismo Genético , RNA Mensageiro/metabolismo
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