Clase 2 Cultivo in vitro

Micropropagation Techniques and Concepts

Introduction to Micropropagation

• The discussion begins with the concept of micropropagation, emphasizing the transition from disorganized tissues to organized tissues for propagation purposes.

• Toti potency in cells allows for the production of organized tissues like embryos from disorganized ones such as callus, depending on propagation goals.

Objectives of Micropropagation

• The primary goal is to propagate clonal plants, utilizing axillary buds or meristems as starting materials due to their high toti potency.

• Morphogenesis can occur through stem differentiation or nodal segments, leading to somatic embryo formation, which is a complex process requiring separate focus.

Steps in Plant Formation

• Initial steps involve forming shoots (caulogenesis), followed by adventitious root formation to create a complete functional plant.

• Starting material for in vitro culture is referred to as explant; it can be leaves, stems, or nodal segments based on the desired propagation method.

Challenges in In Vitro Cultivation

• While theoretically all plants possess toti potency, practical challenges arise with recalcitrant species that struggle with morphogenesis and may produce phenolic compounds that hinder growth.

• Older tissues lose toti potency over time; thus, younger explants are preferred for successful in vitro initiation.

Establishing Aseptic Conditions

• Aseptic conditions are crucial; contaminated starting materials can lead to failure. Common disinfectants include alcohol and hydrogen peroxide.

• Sterilization equipment like autoclaves and laminar flow hoods are essential for maintaining sterile environments during tissue culture processes.

Cultivation Environment and Conditions

Importance of Controlled Environments

• Autoclaves sterilize tools and media used in tissue culture by generating high-pressure steam that kills contaminants.

• Work within laminar flow hoods ensures a sterile air environment while handling plant materials to prevent contamination.

Humidity and Light Considerations

• In vitro conditions often maintain near 100% humidity but provide low light levels (around 50 µmol/m²/s), which can affect plant functionality when transferred outside these conditions.

Transitioning Plants from In Vitro to Ex Situ Conditions

• The transition from controlled environments (in vitro) to natural settings poses significant mortality risks due to inadequate adaptation mechanisms developed under constant humidity.

Growth Phases in Tissue Culture

Growth Dynamics

• Growth phases include lag phase (initial adjustment), exponential growth phase (rapid increase), linear growth phase (steady rate), stationary phase (growth plateaus), and eventual decline if not managed properly.

Subculturing Techniques

• Subculturing involves propagating existing cultures into new media. This technique allows for increased cloning efficiency by taking microshoot cuttings from established cultures.

Conclusion: Practical Applications of Micropropagation

Cloning Efficiency

• Each cutting taken during subculturing results in clones of the original plant material, allowing rapid multiplication compared to traditional methods.

Cultivo In Vitro: Composición y Cambios en el Medio de Cultivo

Cambios en el Medio de Cultivo

• El medio de cultivo se puede refrescar cada 20 a 30 días, permitiendo cambios en su composición básica según el proceso deseado.

• La transformación de micro tallos en plantas completas requiere un cambio significativo en la composición del medio, especialmente en las hormonas.

• La formación de un callo a partir de una hoja implica un medio específico que cambia al buscar multiplicar o diferenciar ese callo.

• Las hormonas son cruciales para determinar la ruta morfogénica que se desea seguir durante el cultivo in vitro.

Historia del Cultivo In Vitro

• Murashige (1974) fue pionero en el desarrollo del cultivo in vitro y creó el medio MS, fundamental para este tipo de cultivos.

• Existen diferentes medios como MS, BBM y B5, que pueden ser modificados según las necesidades hormonales específicas.

Fases del Cultivo In Vitro

Fases Iniciales

• Se describen tres fases principales: iniciación del cultivo (establecimiento aséptico), aumento de propágulos (multiplicación), y preparación para transferencia a condiciones ex vitro.

• La fase de multiplicación incluye subcultivos para inducir la producción masiva de tallos o embriones somáticos.

Preparación para Transferencia

• Es esencial promover actividad fotoautotrófica y elongación antes de transferir las plantas a condiciones ex vitro.

• Las plantas cultivadas in vitro no realizan fotosíntesis adecuadamente debido a la presencia constante de azúcares en el medio.

Selección y Preparación del Material Vegetal

Proceso Adicional

• Se han añadido fases como la selección y preparación del material madre, así como transferencias a condiciones externas controladas.

Importancia del Microambiente

• El microambiente dentro del envase influye significativamente en las características morfológicas y funcionales de las plantas producidas.

Vías de Propagación

Métodos Directos e Indirectos

• Se pueden utilizar métodos directos e indirectos para la propagación, incluyendo brotes adventicios o embriones somáticos.

Ejemplos Prácticos

• En especies amenazadas, se puede iniciar cultivos desde semillas con cruzas controladas o embriones específicos.

Morfogénesis Directa e Indirecta

Diferenciación Celular

• A partir de tejidos somáticos como hojas o raíces se puede generar morfogénesis directa; esto incluye diferenciaciones hacia tallos o embriones somáticos.

Embriogénesis Indirecta

• La embriogénesis indirecta permite formar callos que luego pueden diferenciarse nuevamente en estructuras vegetativas completas.

Transición a Condiciones Ex Vitro

Estrategias para Enraizamiento

• Para llevar micro tallos in vitro al exterior es necesario inducir raíces mediante medios específicos o condiciones controladas.

Consideraciones Críticas

• Un cambio abrupto al ambiente externo sin un adecuado proceso previo puede resultar fatal para las plántulas.

Estudios sobre Micropropagación

Investigación Aplicada

• Se presentan estudios sobre micropropagación efectiva utilizando pistachos, destacando los desafíos económicos asociados con el cultivo in vitro frente al ex vitro.

Coste vs Beneficio

• El costo elevado del cultivo in vitro justifica su uso solo si hay ventajas significativas sobre métodos tradicionales.

Protocolo Experimental

• Los investigadores utilizaron segmentos nodales obtenidos a partir de plántulas escépticas cultivadas bajo condiciones controladas.

Resultados Prometedores

• Los resultados mostraron éxito al cultivar pistachos mediante técnicas específicas adaptadas al contexto económico y biológico requerido.

Vitrification in In Vitro Plant Cultivation

Understanding Vitrification

• Vitrification refers to the appearance of plants that seem shiny and brittle, indicating morphological deficiencies in in vitro plants.

• This condition makes the plants fragile, leading to a higher risk of death due to lack of oxygenation when submerged in liquid media.

Liquid Media Challenges

• Continuous immersion in liquid media can lead to plant mortality due to insufficient oxygen; thus, temporary immersion methods are often preferred.

• Agitation is necessary for submerged cultures to prevent stagnation and ensure adequate oxygen supply.

Components of Culture Media

General Composition

• The composition of culture media depends on the morphogenic pathway desired, including genetic makeup and environmental factors.

• Two types of environments affect morphogenic responses: the internal environment within containers and the growth chamber conditions.

Nutrient Delivery

• Liquid media are less effective as physical supports for plant growth compared to solid media; they are better suited for callus production rather than microshoot generation.

• Macronutrients (nitrogen, potassium, calcium, magnesium, phosphorus, sulfur) and micronutrients (iron, molybdenum, copper, boron, zinc) play crucial roles in plant metabolism.

Stock Solutions Preparation

Efficient Media Preparation

• To streamline preparation processes, stock solutions are created for macronutrients and micronutrients instead of adding each component individually.

• Stocks allow for concentrated nutrient solutions that simplify medium preparation across various scales from research labs to commercial production.

Organic Components

• Vitamins (e.g., thiamine and myo-inositol), amino acids (e.g., glycine), and other organic components serve as essential nutrients but their specific roles remain largely unexplored.

Role of Sugars in Plant Growth

Carbon Sources

• Sugars like sucrose provide energy but can hinder autotrophy by reducing chloroplast development and limiting CO2 availability during cultivation.

• Higher sugar concentrations may enhance growth rates but also increase vitrification risks; lower concentrations mitigate these issues at the cost of slower growth.

Gel Agents in Culture Media

Importance of Gel Agents

• Gel agents must be inert and able to withstand sterilization processes. Common agents include agarose or proprietary gels like Gellan gum which offer visibility into contamination levels.

pH Adjustments

• Maintaining an optimal pH (around 6.2 - 5.8 range recommended for nutrient availability); deviations can affect gel consistency and nutrient absorption efficiency.

Finalizing Culture Medium Preparation

Comprehensive Medium Formulation

• The basal medium consists of concentrated stocks combined with sugars and hormones tailored according to specific research needs or species requirements.

Sterilization Techniques

Autoclaving is critical for preventing contamination; however, it may degrade sensitive components like vitamins if not managed properly. Alternative sterilization methods exist but have limitations regarding germination efficacy.