Abstract: The following 8F1, hybrid cultivars of broccoli were employed:Green Charger, Green Valient (these two cultivars came from Japan) :Futura，Skiff, Naptune, Inwaprium, Corvet and Atlantic (these six cultivars came from Germany).The leaves of propagated plantlets in vitro were cut into 1-2 mm pieces with a wash solution(a) for plasmolysis, then were incubated in 10ml enzyme solution(b)，and were placed on a rotary shaker(50 rpm)under light(2500 lux)at 21°c for 3 hours.The mixture was filtered through a 45 μm nylon screen (or stainless steel net) into centrifuge tubes and centrifuged 5 min at 100×g.The pellet was resuspended in 3.5ml 0.5M sucrose solution(c) and 1.0 ml wash solution(a) was layered on the top and the tubes centrifuged at 100×g for 5 min.The floating green band of protoplasts between the sucrose solution and the wash solution was carefully removed, then was resuspended in 10 ml wash solution (a) and centrifuged at 100×g for 7 min.The protoplasts were resuspended in 0.5- 1 ml wash solution (a) and dripped on to the drops of warm agarose(i)in 3cm plastic petri dishes, The agarose drops were solidified at once with a cold ironplate. 2-3ml liquid medium (d or f) was added in to dished around the agarose drops so that the agarose drops were floated.The petri dishes were sealed with paraffin film, These petri dishes were placed in a transparent plastic box (20×20×7cm) with close lid and together with two petri dishes(5 cm in diameter)，which were full of distilled water to maintain humidity，then were incubated in the culture-room at 25°C under light (4000 lux,16h).On the 7th day after isolation,1.5ml liquid medium was removed from each dish to a new one, and 1.5 ml fresh medium with reduced concentration of mannitol(c) was added to each dish.3-5 days after isolation of protoplasts, the new cell wall formation and the first cell division were found, On the 7th day after isolation,there were 4一8 colonies.M ost of the colonies had a ball shape.But some of them showed a peculiar form, which,both in size and form，were much different from the ball shape colonies.3-5 weeks after isolation,some p-calli developed, When notably large (2-3mm in diameter, usually 6-3 weeks after isolation)，these p-calli were transferred to medium Girmen C (g) with 8% agar solidified.Once calluses were 1 cm in diameter,they were individually transferred to agar一solified medium and shoots from these Girmen G (h).5months after isolation some roots calluses were regenerated.We have obtained cell division，celled colonies and p-calli in Green charger，Green Valient and Naptunc，calluses in Skiff and Futura,roots and shoots regeneration in Futura. The solutions and media are listed as follows: (mg/l) (a)Wash solution; 1.00 MES+100 CaCI}+100 NaHz PO;+0, 3 M mannitol，pH 5.5一5.8 (b)Enzyme solution:2%cellulase+1%macerase in 10 ml solution(a)，pH6.8 (c) Sucrose solution:0,5 M sucrose+5 m M MES, pH5,5一5,8 (d) WT19八medium; basic medium T+0.15M sucrose+0.25M mannitol+2.4-D1+NAA 1+BAP0.5, pH 5.5-5.8, filter sterilized (e) WT29/1 medium: basic madium T+0.15 sucrose+0.075M mannitol+2,4-NAA1+BAP0.5,pH5.5-5.3,filter sterilized (f)Girmen 3 medium:Girmen basic medium+0.4M glucose+2,4-D1+NAA1+BAP0.5,pH5.5-5.8,filter sterilized (g)Girmen C medium:Girmen basic medium+31.2g/L sucrose+2,4-D0.1+NAA1+BAP0.5,pH5.5-5.8,filter sterilized (h)Girmen G medium:Girmen basic medium+10g/L sucrose+IAA0.1=BAP0.5+8g/L agar powder,pH5.5-5.8 (i)Agarose:1% agarose in solution(a)

Key words: Protoplasts culture, Shoots regeneration, Broccoli