RESUMO
Temporary immersion systems (TIS) have been widely recognized as a promising technology for micropropagation of various plant species. The TIS provides a suitable environment for culture and allows intermittent contact of the explant with the culture medium at different immersion frequencies and aeration of the culture in each cycle. The frequency or immersion is one of the most critical parameters for the efficiency of these systems. The design, media volume, and container capacity substantially improve cultivation efficiency. Different TIS have been developed and successfully applied to micropropagation in various in vitro systems, such as sprout proliferation, microcuttings, and somatic embryos. TIS increases multiplication and conversion rates to plants and a better response during the ex vitro acclimatization phase. This article covers the use of different immersion systems and their applications in plant biotechnology, particularly in plant tissue culture, as well as its use in the massive propagation of plants of agroeconomic interest.
Assuntos
Aclimatação , Desenvolvimento Vegetal , Meios de Cultura/química , Técnicas de Cultura de Tecidos/métodos , Técnicas de Cultura de Tecidos/instrumentação , Brotos de Planta/crescimento & desenvolvimento , Brotos de Planta/fisiologia , Plantas , Imersão , Técnicas de Embriogênese Somática de Plantas/métodosRESUMO
In this chapter, we report advances in tissue culture applied to Passiflora. We present reproducible protocols for somatic embryogenesis, endosperm-derived triploid production, and genetic transformation for such species knowledge generated by our research team and collaborators in the last 20 years. Our research group has pioneered the work on passion fruit somatic embryogenesis, and we directed efforts to characterize several aspects of this morphogenic pathway. Furthermore, we expanded the possibilities of understanding the molecular mechanism related to developmental phase transitions of Passiflora edulis Sims. and P. cincinnata Mast., and a transformation protocol is presented for the overexpression of microRNA156.
Assuntos
Passiflora , Técnicas de Embriogênese Somática de Plantas , Técnicas de Cultura de Tecidos , Passiflora/genética , Passiflora/crescimento & desenvolvimento , Técnicas de Embriogênese Somática de Plantas/métodos , Técnicas de Cultura de Tecidos/métodos , Transformação Genética , MicroRNAs/genética , Plantas Geneticamente Modificadas/genética , Plantas Geneticamente Modificadas/crescimento & desenvolvimento , Endosperma/genética , Endosperma/crescimento & desenvolvimento , Regulação da Expressão Gênica de PlantasRESUMO
The coconut tree is a crop widely distributed in more than 90 countries worldwide. It has a high economic value derived from the large number of products obtained from the plant, with fast-growing global markets for some of them. Unfortunately, coconut production is decreasing mainly due to the old age of the plants and devastating pests and diseases, such as phytoplasma disease lethal yellowing (LY). Massive replanting is required with phytoplasma-resistant and high-yielding selected coconut plants to keep up with the market demand for fruit. For this purpose, an efficient micropropagation technology via somatic embryogenesis has been established at CICY, yielding fully developed vitro-plants grown within an in vitro environment. Hence, the last stage of the micropropagation process is the acclimatization of the vitro-plants, which are gradually adapted to live in external conditions outside the glass container and the growth room. A protocol has been developed at CICY to acclimate the coconut vitro-plants, and close to 80% survival can be obtained. This protocol is described here.
Assuntos
Aclimatação , Cocos , Técnicas de Embriogênese Somática de Plantas/métodos , PhytoplasmaRESUMO
Somatic embryogenesis (SE) is a clear example of cellular totipotency. The SE of the genus Coffea has become a model for in vitro propagation for woody species and for the large-scale production of disease-free plants that provide an advantage for modern agriculture. Temporary immersion systems (TIS) are in high demand for the propagation of plants. The success of this type of bioreactor is based on the alternating cycles of immersion of the plant material in the culture medium, usually a few minutes, and the permanence outside the medium of the tissues for several hours. Some bioreactors are very efficient for propagating one species but not another. The efficiency of bioreactors depends on the species, the tissue used to propagate, the species' nutritional needs, the amount of ethylene produced by the tissue, and many more. In this protocol, we show how we produce C. canephora plants that are being taken to the field.
Assuntos
Coffea , Técnicas de Embriogênese Somática de Plantas , Técnicas de Embriogênese Somática de Plantas/métodos , Coffea/crescimento & desenvolvimento , Coffea/genética , Reatores Biológicos , Sementes/crescimento & desenvolvimento , Meios de Cultura/químicaRESUMO
Over the years, our team has dedicated significant efforts to studying a unique natural dye-producing species, annatto (Bixa orellana L.). We have amassed knowledge and established foundations that support the applications of gene expression analysis in comprehending in vitro morphogenic regeneration processes, phase transition aspects, and bixin biosynthesis. Additionally, we have conducted gene editing associated with these processes. The advancements in this field are expected to enhance breeding practices and contribute to the overall improvement of this significant woody species. Here, we present a step-by-step protocol based on somatic embryogenesis and an optimized transformation protocol utilizing Agrobacterium tumefaciens.
Assuntos
Agrobacterium tumefaciens , Bixaceae , Transformação Genética , Agrobacterium tumefaciens/genética , Bixaceae/genética , Bixaceae/metabolismo , Técnicas de Cultura de Tecidos/métodos , Técnicas de Embriogênese Somática de Plantas/métodos , Edição de Genes/métodos , Plantas Geneticamente Modificadas/genética , Plantas Geneticamente Modificadas/crescimento & desenvolvimentoRESUMO
This chapter presents an efficient protocol for regenerating Carica papaya plants via somatic embryogenesis from immature zygotic embryos from economically important papaya genotypes. To achieve regenerated plants from somatic embryos, in the present protocol, four induction cycles are required, followed by one multiplication cycle and one regeneration cycle. With this optimized protocol, 80% of somatic embryos can be obtained in only 3.5 months. At this stage, calli containing more than 50% globular structures can be used for transformation (via agrobacterium, biobalistics, or any other transformation method). Once transformed, calli can be transferred to the following steps (multiplication, elongation, maturation, rooting, and ex vitro acclimatization) to regenerate a transformed somatic embryo-derived full plant.
Assuntos
Carica , Genótipo , Técnicas de Embriogênese Somática de Plantas , Carica/genética , Carica/embriologia , Técnicas de Embriogênese Somática de Plantas/métodos , Transformação Genética , Plantas Geneticamente Modificadas/genética , Regeneração/genética , Sementes/genética , Sementes/crescimento & desenvolvimentoRESUMO
Omic tools have changed the way of doing research in experimental biology. The somatic embryogenesis (SE) study has not been immune to this benefit. The transcriptomic tools have been used to compare the genes expressed during the induction of SE with the genes expressed in zygotic embryogenesis or to compare the development of the different stages embryos go through. It has also been used to compare the expression of genes during the development of calli from which SE is induced, as well as many other applications. The protocol described here is employed in our laboratory to extract RNA and generate several transcriptomes for the study of SE on Coffea canephora.
Assuntos
Coffea , Perfilação da Expressão Gênica , Regulação da Expressão Gênica de Plantas , Técnicas de Embriogênese Somática de Plantas , Transcriptoma , Coffea/genética , Coffea/embriologia , Coffea/crescimento & desenvolvimento , Técnicas de Embriogênese Somática de Plantas/métodos , Perfilação da Expressão Gênica/métodos , Transcriptoma/genética , Sementes/genética , Sementes/crescimento & desenvolvimento , Regulação da Expressão Gênica no DesenvolvimentoRESUMO
Plant cell, tissue, and organ cultures (PCTOC) have been used as experimental systems in basic research, allowing gene function demonstration through gene overexpression or repression and investigating the processes involved in embryogenesis and organogenesis or those related to the potential production of secondary metabolites, among others. On the other hand, PCTOC has also been applied at the commercial level for the vegetative multiplication (micropropagation) of diverse plant species, mainly ornamentals but also horticultural crops such as potato or fruit and tree species, and to produce high-quality disease-free plants. Moreover, PCTOC protocols are important auxiliary systems in crop breeding crops to generate pure lines (homozygous) to produce hybrids for the obtention of polyploid plants with higher yields or better performance. PCTOC has been utilized to preserve and conserve the germplasm of different crops or threatened species. Plant genetic improvement through genetic engineering and genome editing has been only possible thanks to the establishment of efficient in vitro plant regeneration protocols. Different companies currently focus on commercializing plant secondary metabolites with interesting biological activities using in vitro PCTOC. The impact of omics on PCTOC is discussed.
Assuntos
Células Vegetais , Técnicas de Cultura de Tecidos , Técnicas de Cultura de Células/métodos , Produtos Agrícolas/genética , Produtos Agrícolas/crescimento & desenvolvimento , Melhoramento Vegetal/métodos , Células Vegetais/metabolismo , Desenvolvimento Vegetal/genética , Plantas/genética , Plantas/metabolismo , Técnicas de Cultura de Tecidos/métodosRESUMO
Gamma radiation (60Co)-induced mutagenesis offers an alternative to develop rice lines by accelerating the spontaneous mutation process and increasing the pool of allelic variants available for breeding. Ionizing radiation works by direct or indirect damage to DNA and subsequent mutations. The technique can take advantage of in vitro protocols to optimize resources and accelerate the development of traits. This is achieved by exposing mutants to a selection agent of interest in controlled conditions and evaluating large numbers of plants in reduced areas. This chapter describes the protocol for establishing gamma radiation dosimetry and in vitro protocols for optimization at the laboratory level using seeds as the starting material, followed by embryogenic cell cultures, somatic embryogenesis, and regeneration. The final product of the protocol is a genetically homogeneous population of Oryza sativa that can be evaluated for breeding against abiotic and biotic stresses.
Assuntos
Raios gama , Mutagênese , Oryza , Sementes , Oryza/genética , Oryza/efeitos da radiação , Oryza/crescimento & desenvolvimento , Mutagênese/efeitos da radiação , Sementes/genética , Sementes/efeitos da radiação , Sementes/crescimento & desenvolvimento , Regeneração/genética , Técnicas de Embriogênese Somática de Plantas/métodosRESUMO
Somatic embryogenesis in Agave genus has been induced; however, it is desirable to increase the rate of growth to get a more efficient propagation system. In this chapter, we present in detailed a protocol for somatic embryogenesis in Agave cupreata and the use of silver nanoparticles in a temporary immersion system. This is an efficient method that can be used commercially to improve the production and germination of somatic embryos.