Somatic embryogenesis and plant regeneration in Medicago arborea L.

Abstract

An efficient plant regeneration system employing cotyledons, hypocotyls, petioles and leaves as explants and characterized by continuous and prolific production of somatic embryos, has been developed with Medicago arborea ssp. arborea. The optimal somatic embryogenic response was obtained using a two-step protocol, where explants were incubated under a 16 h photoperiod for 2 mo. on Murashige and Skoog (MS) medium containing 2,4-dichlorophenoxyacetic acid (2,4-D; 9 mM) and kinetin (9 mM), and followed by transfer to kinetin-free MS medium with 2,4-D (2.25 mM). Removal of the cytokinin and a reduction in the concentration of auxin (2.25 mM) in the second step of culture were critical for enhanced production of somatic embryos. The best explants proved to be cotyledons and petioles (i.e. a mean of 18X0^0X70 somatic embryos at 3 mo. for petiole culture). Somatic embryos were converted into normal plantlets 8X00 X89% when cultured on basal MS medium with 5 mM indolebutyric acid. No somatic embryos were obtained when thidiazuron was used in the culture media. Using petioles as explants and N 6 -benzyladenine (BA), embryogenesis was induced in the second step of culture when BA was removed from the medium and the concentration of 2,4-D was decreased to 2.25 mM.

In vitro Culture of Medicago arborea L. Anthers: Initial Response

“…To check the viability of the embryos, their germination capacity was determined after separating them from the calli and culturing for 10 days on basal MS medium (Murashige and Skoog 1962) with indolebutyric acid (IBA) (5 lM), as described by Gallego et al (2001).…”

“…To regenerate plantlets, the method of Gallego et al (2001) was followed. After 10 days of culture in MS media plus IBA, embryos were transferred to small pots containing MS with half the salt concentration and no sucrose.…”

“…During this time, the parafilm was gradually removed to favour the acclimatisation the cultures to atmospheric conditions. Subsequently, the seedlings were transferred to sterile soil and, after 2 weeks of growth, they were transferred to pots with soil to complete their development (Gallego et al 2001). Leaves of these plants were collected for further analysis.…”

“…This is a bushy Mediterranean legume that is of considerable agronomic interest because it is resistant to drought and has seeds that do not exhibit a dormancy stage at any time of the year (Lesins and Lesins 1979;Gallego et al 2001).…”

“…In vitro culture techniques offer many possibilities for the rapid propagation of M. arborea (Gallego et al 2001). Peng and Wolyn (1999) reported that one of the most important biotechnological developments in recent years for optimising the production of new plant varieties was the possibility of obtaining dihaploid plants (Lee et al 2003).…”