Embriogenesis

Medicago truncatula handbook

version November 2006

Cell suspension cultures
A. Iantcheva1*, M. Vlahova1, A. Atanassov1, A. S. Duque Santos 2, P. Fevereiro 2, 3
2**

, S. Araújo 2**, D. F. dos

1.

AgroBioInstitute, 1164 Sofia, Bul. Dragan Tzankov 8, Bulgaria

* Corresponding author aneliaiancheva@abi.bg ; anelia64mimi@yahoo.com
2.

Laboratory of Plant Cell Biotechnology,Instituto de Tecnologia Química e Biológica (ITQB), Apt. 127, 2781-901 Oeiras, Portugal
3.

Departamento de Biologia Vegetal, Faculdade de Ciências da Universidade de Lisboa, Campo Grande, 1749-016 Lisboa, Portugal
**

Corresponding authors: sduque@itqb.unl.pt; saraujo@itqb.unl.pt

Table of contents 1. 2. 3. 4. 5. Introduction Cell suspension culture of M. truncatula cv. R 108 1 initiated fromleaf and root explants Long-term embryogenic cell suspension cultures of Medicago truncatula cv. Jemalong line M9-10a References Figures and Tables

1. Introduction Somatic embryogenesis is the process by which the somatic cells give rise to bipolar structure, which develop to whole plants without gamete fusion. It is strongly depends on the plant genotype, type and physiological state of theexplants, the composition of the culture medium and the conditions of cultivation. Somatic embryogenesis in Medicago truncatula is strongly genotype-dependent. The process could be direct when the embryogenic structures develop directly from the initial explant, or indirect through a callus stage. Regeneration via direct somatic embryogenesis in liquid media for M. truncatula line R 108 1 from leafand root explant has been established (Iantcheva et al. 2001; Iantcheva et al. 2005). These procedures based on suspension culture for regeneration of this model species offer new advantages: omit callus stage that allow shortening of the process of somatic embryogenesis and provide basis for morphological, biochemical and molecular studies of the nature of somatic embryogenesis from single cell towhole plant.

Cell suspension cultures

page 1 of 12

Medicago truncatula handbook

version November 2006

In Medicago truncatula cv. Jemalong only few selected lines/genotypes are described to have embryogenic capacity. A highly embryogenic line (M9-10a) was obtained via somatic embryogenesis from a line with very low embryogenic potential (Neves et al., 1999). Although an efficientmethod for transformation-regeneration through somatic embryogenesis using leaf explants have been established for this M9-10a Jemalong line (Araújo et al., 2004) an alternative embryogenic cell suspension culture protocol represents an in vitro attractive system for mutant selection, mass propagation, and gene transfer experiments. Using the method here described long-term cell suspension culturesof M. truncatula line M9-10a were obtained and plants successfully regenerated via somatic embryogenesis. Constant availability of embryogenic-competent cells, the possibility of scaling-up and the reduction of time needed for plant regeneration are advantages of this protocol. 2. Cell suspension culture of M. truncatula cv. R 108 1 initiated from leaf and root explants A. Iantcheva*, M. Vlahova,A. Atanassov AgroBioInstitute, 1164 Sofia, Bul. Dragan Tzankov 8, Bulgaria * Corresponding author aneliaiancheva@abi.bg ; anelia64mimi@yahoo.com Plant material For initiation of suspension culture plant explants from 30 days old in vitro plant material from Medicago trunactula line R 108 1 established from seeds are used. The seeds of line R 108 1 were kindly provided by Institut des Sciences duVégétale, Gif-sur Yvette, France where it was established (Hoffman et al. 1997). They are rinsed with water prior surface sterilization with 6% solution of sodium hypochlorite (commercial bleach) for 15 min. and then rinsed three times with sterile distilled water. Seedlings developed on MS (Murashige and Skoog, 1962) basal medium are propagating via cuttings. All plant material is cultivated in...