Efficient Plant Regeneration from Alfalfa Callus by Osmotic Stress Treatment

ABSTRACT Effects of culture medium supplements and osmotic stress treatment on embryogenic callus induction andsomatic embryogenesis were investigated in order to optimize tissue culture conditions of alfalfa( Medicagosativa L.). SH medium containing 5mg/L 2,4-D and 0.2mg/L kinetin was optimal for embryogenic callusinduction from cotyledon tissue of alfalfa. Somatic embryos were formed when the embryogenic callus wascultured on SH medium supplemented with 1mg/L 2,4-D and 2mg/L BA. Supplementation of 5mM L-pro-line and 1g/L casein hydrolysate into the regeneration medium further increased plant regenerationfrequency. Osmotic stress treatment of callus appeared to improve the frequency of somatic embryo for-mation, but the frequency of somatic embryo formation differed by the osmotic stress treatment using dif-ferent osmotic stressors. The highest plant regeneration frequency of 30.7% was observed when embryogeniccallus was treated with 0.7M sucrose for 18h. Efficient regeneration system established in this study will beuseful for molecular breeding of alfalfa through genetic transformation.

[1]  P. B. K. Kishor,et al.  Enhanced plant regeneration in grain and sweet sorghum by asparagine, proline and cefotaxime , 2005, Plant Cell Reports.

[2]  S. Eapen,et al.  Influence of phytohormones, carbohydrates, aminoacids, growth supplements and antibiotics on somatic embryogenesis and plant differentiation in finger millet , 1990, Plant Cell, Tissue and Organ Culture.

[3]  Itziar Lanas,et al.  Improvement of somatic embryogenesis in Medicago arborea , 2004, Plant Cell, Tissue and Organ Culture.

[4]  B. G. Thompson,et al.  Genotypic effects on somatic embryognesis and plant regeneration from callus cultures of alfalfa , 2004, Plant Cell, Tissue and Organ Culture.

[5]  Mikihisa Umehara,et al.  Stress-induced somatic embryogenesis in vegetative tissues of Arabidopsis thaliana. , 2003, The Plant journal : for cell and molecular biology.

[6]  R. Dixon,et al.  Legume Natural Products: Understanding and Manipulating Complex Pathways for Human and Animal Health1 , 2003, Plant Physiology.

[7]  José Ignacio Cubero Salmerón.  Biotechnology and the Improvement of Forage Legumes , 2002 .

[8]  P. Gallego,et al.  Somatic embryogenesis and plant regeneration in Medicago arborea L. , 2001, In Vitro Cellular & Developmental Biology - Plant.

[9]  W. McNabb,et al.  The implications of condensed tannins on the nutritive value of temperate forages fed to ruminants , 1999, British Journal of Nutrition.

[10]  P. Bauer,et al.  Rapid and efficient transformation of diploid Medicago truncatula and Medicago sativa ssp. falcata lines improved in somatic embryogenesis , 1998, Plant Cell Reports.

[11]  P. Morris.  Manipulating condensed tannins in forage legumes , 1997 .

[12]  Daniel C. W. Brown,et al.  Biotechnology and the improvement of forage legumes , 1997 .

[13]  M. R. Koupai-Abyazani,et al.  Purification and characterization of a proanthocyanidin polymer from seed of alfalfa (Medicago sativa Cv. beaver) , 1993 .

[14]  H. Kamada,et al.  Induction of Somatic Embryogenesis in Carrot by Osmotic Stress , 1993 .

[15]  T. Kiyosue,et al.  Induction of somatic embryogenesis in carrot by heavy metal ions , 1990 .

[16]  Daniel C. W. Brown,et al.  A novel system for rapid high frequency somatic embryogenesis in Medicago sativa , 1987 .

[17]  D. Skinner,et al.  Regeneration of alfalfa plants from protoplasts of selected Regen S clones , 1981 .

[18]  M. Davey,et al.  Organogenesis and somatic embryogenesis in tissues derived from leaf protoplasts and leaf expiants of Medicago sativa , 1980 .

[19]  J. Saunders,et al.  Production of Alfalfa Plants from Callus Tissue1 , 1972 .

[20]  R. U. Schenk,et al.  Medium and techniques for induction and growth of monocotyledonous and dicotyledonous plant cell cultures , 1972 .