Enhancement by Osmotic Treatment of Hairy Root Transformation of Alfalfa Suspension Cultures, and Chromosomal Variation in the Transformed Tissues

Abstract

Seeds of alfalfa were germinated on Murashige and Skoog medium without phytohormones. The hypocotyls ofseedlings were excised and cultured on the same medium with 2,4- dichlorophenoxyacetic acid (9.05 mM) to induce callus. Granular calluses were suspended and cultured in Schenk and Hildebrandt medium supplemented with 2.26 mM 2,4-dichlorophenoxyacetic acid on a shaker at 80 rpm. Alfalfa suspension cultures were treated with 0.45 M mannitol for 1 h, and washed twice with 0.16 M CaCl₂ .2H₂ O. After pretreatment, the pellets were resuspended in Schenk and Hildebrandt medium without phytohormone (10 mL g^-1 suspension cultures), and 0.2 mL of Agrobacterium rhizogenes suspension was added. A mixture of alfalfa cells and Agrobacterium was co-cultured at 25 ± 2˚C for 2 days. Transformants (transformed calluses and hairy roots) were obtained by hormone-free selection. Several factors, such as culturing stage of suspension cultures, phytohormone constitution of suspension medium and basal selection medium of transformants, affected the transformation frequency remarkably. Paper electrophoresis revealed that over 70% of transformants could synthesise agropine and mannopine. A comparative cytological analysis revealed the number and structural alterations of chromosomes in the resulting materials. The transformed tissue was not able to be regenerated, possibly due to chromosomal abnormalities.