Selecting the Electrofusion Condition of Alfalfa Protoplast

The somatic hybridization of asymmetric fusion between protoplasts isolated from callus of Russian variegated alfalfa and 'Gannong No.4' alfalfa was obtained through electrofusion method.The effects of different inactivation pretreatments,electric field conditions and protoplast densities on alfalfa protoplast fusion were studied.Test results showed that the optimum inactivation pretreatments were that Russian variegated alfalfa protoplast was treated with ultraviolet radiation by 5 min and 'Gannong No.4' alfalfa protoplast was treated with 6 mmol·L-1 iodoacetamide.The optimum electrofusion parameters were AC electric field intensity for 15~20 V·cm-1,frequency for 2000~2500 kHz,DC electric field intensity for 200~250 V·cm-1,pulse width for 40 μs and pulse 3 times.The optimum protoplast density is 3×105~5×105 mL-1.The highest protoplast binary fusion frequency reached 13.8% with above conditions.