Author: Li Chang-gong, Zhou Chang and Yang Hong-yuan
J Integr Plant Biol 1994, 36 (12): -.
It has been reported that "gameto-somatic hybridization" was induced by fusion of microspore tetrad protoplasts with somatic protoplasts in Nicotiana and Petunia. However, since the success of isolation of pollen protoplasts in recent years, the use of protoplasts at pollen stage as one of the fusion partners in such hybridization is a novel experimentation. Young pollen protoplasts were isolated from the pollen grains of Brassica chinensis at mid-late unicellular to early bicellular stage the pollens for 1.5--2.5 h at 25℃ in a CPW solution containing 0.8 % of eellulase, 0.5 % pectinase, 0.1% pectolyase, 1 3 % mannitol, 1 0 % glucose, 0. 3% potassium dextran sulphate and 3 mmol/L MES. The purified pollen protoplasts were then fused with the hypocotyl protoplasts of B. napus by PEG method. Heterokaryons were identified by means of visualization of the fluorescence from FITC-prela-beled pollen protoplasts. In order to increase heterokaryons and reduce hypocotyls homokaryons, the denstity of hypocotyl protoplasts were lowered and the ratio of the number of hypocotyl vs. pollen protoplasts were adjusted from 1 : 3 to 1 : 6. The fusion products were cultured in a liquid KM8p medium supplemented with 0.4 mol/L glucose, 0.8 mg/L 2, 4-D, 0.25 mg/L NAA. 0. 5 mg/L BA, 500 mg/L glutamine and 3 mmol/L MES where cell division and callus formation took place. The calli, after being transferred to a MS medium supplemented with 2.0 mg/L BA, 3 % sucrose and 0.4 % agarose, differentiated into a few shoots. The shoots were transferred onto a half-strength MS medium supplemented with 2% sucrose, 0.1--0. 2 mg/L NAA, 0.5 mg/L IBA and 20% potato juice for root formation. Finally, three plantlets were regenerated. Chromosome counts by roottip squash method revealed that one plantlet was 2n= 48, corresponding to an allotriploid resulted from a fusion between one pollen protoplast of B. chinensis (2n = 20) and one hypocotyl protoplast of B. napus (2n = 38), and the other two plantlets were 2n = 58, which might be an allotetraploid originated from a fusion between two pollen protoplasts and one hypocotyl protoplast. The isozyme patterns of leaf esterases showed that all the three plantlets had bands characteristic of both parents. This is the first case of success in "gameto-somatic hybridization" by using pollen protoplasts rather than tetrad protoplasts as the haploid partner.