Laura V. Janeiro

Cold storage of shoot cultures and alginate encapsulation of shoot tips of Camellia japonica L. and Camellia reticulata Lindley

Abstract In vitro shoot cultures of 8 clones or cultivars of Camellia japonica L. and Camellia reticulata Lindley were stored at 2–4 °C for up to 12 months. Survival frequencies near 100% were obtained during the first or second subcultures after cold storage in seven of the eight clones assayed, but the other clone could not be profitably stored for more than 6 months. When shoot tips and nodal expiants of C. japonica L. (clone 2 and cv. Alba Plena) were encapsulated in alginate beads, the regrowth of shoot tips depended on the components of the gel matrix but that of nodal expiants was very poor regardless of the encapsulation conditions. The best morphogenetic response of the shoot tips was obtained when the encapsulating gel was supplemented with the minerals and vitamins of Murashige and Skoogs medium, 3% sucrose and clone-specific growth regulators. The size of the shoot tips used and the concentration of sucrose added to the gel matrix also influenced the capacity for regrowth. Encapsulated shoot tips of C. japonica L. stored at 2–4 °C or 18–20 °C survived for shorter periods of time than unencapsulated shoot tips stored at low temperature: after 75 days of storage, only 10% of encapsulated shoot tips survived at 2–4 °C and 7% at 18–20 °C.

In vitro response of encapsulated somatic embryos of camellia

Somatic embryos of Camellia japonica were hydrogel encapsulated using 3% sodium alginate and 0.1 M calcium chloride to produce synthetic seeds. Both germinability and repetitive embryogenesis capacity of the encapsulated embryos were investigated. The frequency of in vitro germination into plants of artificial seeds was affected by various nutrient additives included in the encapsulating matrix. The addition of Ca-free Murashige and Skoog basal medium plus 3% sucrose plus 14.4 µM gibberellic acid and 28.5 µM indole-3-acetic acid to the alginate capsule resulted in 63% plant recovery rate, which was similar to that of non-encapsulated embryos. Encapsulation of somatic embryos did not negatively affect the maintenance of their embryogenic competence, the mean number of secondary embryos being significantly increased when the alginate beads included the growth regulators of the secondary embryogenesis medium (4.44 µM 6-benzyladenine and 0.41 µM indole-3-butyric acid). Storage at 4°C significantly reduced the survival and germination into plants frequencies of both encapsulated and non-encapsulated embryos, but the reduction was much greater for non-encapsulated embryos. Plant recovery of encapsulated embryos was 40% and 30% following storage for 30 and 60 days, respectively.

Effect of cold storage on somatic embryogenesis systems of Camellia

SummaryThe effects of short-to medium-term cold storage (3-4°C) on the maintenance of embryogenic capacity and the germination and conversion of somatic embryo cultures of Camellia were investigated. Four culture lines maintained by repetitive embryogenesis were used: three C. japonica lines and one C. reticulata cv. Mouchang line. Increasing cold storage time (3, 6, 9, and 12 months), decreased the frequency of surviving embryo clusters and the mean number of surviving embryos per cluster. Eight week old embryogenic cultures were stored for one or two months, after which secondary embryos were isolated from the parent embryos and transferred to the germination medium. Two months of chilling significantly improved plantlet conversion abilities of secondary embryos, with rates ranging from 76–100% (depending on the embryogenic line) against 32–65%, respectively, in the absence of cold treatment. The incidence of secondary embryogenesis during germination also decreased after cold treatment. Cold storage of...

Cryopreservation of somatic embryos and embryonic axes of Camellia japonica L.

SummaryCryopreservation in liquid nitrogen was attempted with both somatic embryos and zygotic embryonic axes of the ornamental Camellia japonica L. Several protective measures were applied to somatic embryos (desiccation, chemical protectors, hardening by culture at low temperatures, encapsulation in alginate beads), but none allowed somatic embryos cultures to survive after 24 h in liquid nitrogen. Embryonic axes, however, were easily cryopreserved by means of the simplest technique: desiccation in a laminar flow hood and direct immersion in liquid nitrogen. Although the causes of the difference in cryopreservability between the two types of material are not known, one might be the difference between their degrees of differentiation and water content.

Conservación del germoplasma de especies leñosas con técnicas de cultivo in vitro y alamacenamiento en frío

El mantenimiento in vitro de tejidos vegetales constituye un procedimiento esencial para la conservacion y el intercambio de recursos geneticos. El cultivo in vitro se define como un proceso en el que las celulas, tejidos, o organos vegetales son cultivadas en condiciones asepticas en un ambiente controlado. Esta metodologia ofrece la posibilidad de almacenar un gran numero de muestras en un espacio reducido, ademas de permitir la conservacion de aquellas especies con semillas de poca viabilidad, cultivos de propagacion clonal en masa, cultivos altamente heterocigoticos o cultivos que requieren ser propagados vegetativamente para conservar su integridad genetica. Los cultivos establecidos in vitro se pueden conservar a corto plazo (desde 1 semana a dos meses) y a medio plazo manipulando las condiciones de crecimiento. La conservacion a largo plazo de cultivos in vitro se puede lograr usando condiciones de almacenamiento en nitrogeno liquido o crioconservacion. El objetivo de este trabajo ha sido describir las principales metodologias para conservacion in vitro a corto y medio plazo de germoplasma de una serie de especies lenosas lo que ha permitido al Departamento de Fisiologia Vegetal del Instituto de Investigaciones Agrobiologicas de Galicia (CSIC) el establecimiento de un banco de germoplasma de especies lenosas.

Biotechnological Approaches for the Improvement and Conservation of Alnus glutinosa (L.) Gaertner

European alder [Alnus glutinosa (L.) Gaertner], also called black alder or European black alder, is a medium-sized tree that is widely distributed throughout Europe. In the few decades, black alder populations have declined drastically, partly as a result of deforestation and the disappearance of riparian habitats but mainly because of alder blight disease, caused by Phytophthora alni. In order to protect these important genetic resources, the existing conservation methods must be improved and new tools developed.

Efecto de los carbohidratos sobre la micropropagación de Alnus glutinosa (L.) Gaertn

En este trabajo se describe el efecto de los carbohidratos sobre el desarrollo de los brotes y la formacion de raices adventicias en dos clones de origen adulto de Alnus glutinosa (L.) Gaertn. previamente establecidos in vitro. Para la multiplicacion se utilizo el medio WPM suplementado con 0,5 mg/l de acido indol acetico, 0,2-0,1 mg/l de benciladenina y 0,5 mg/l de zeatina, como fuente hidrocarbonada se utilizaron glucosa (111 o 166 mM) o sacarosa (58 o 87 mM). El tipo de azucar anadido al medio de cultivo tuvo una notable influencia sobre el desarrollo de los brotes, obteniendo los mejores resultados cuando se utilizo glucosa 111 mM. Con la concentracion mas elevada de glucosa (166 mM) los brotes presentaban signos de hiperhidricidad. Los mejores porcentajes de enraizamiento se obtuvieron al combinar el efecto de la glucosa y el tratamiento con acido indol butirico 0,1 mg/l durante los 7 primeros dias. Las plantas obtenidas in vitro se transfirieron al suelo con porcentajes de supervivencia superiores al 90%.