嫩茎花椰菜原生质体培养和新枝再生

The Protoplasts Isolation, Culture and Shoots Regeneration of Broccoli

研究了8个杂种一代嫩茎花椰菜品种,在5个品种中得到了细胞分裂、多细胞团和愈伤组织;1个品种的愈伤组织再生了新枝。离体繁殖的无菌苗叶片剪成细条,酶液由2％纤维素酶和1％解析酶组成,21℃、50转/分摇床上分离3小时。0.5M蔗糖液漂浮纯化。接种于1％琼脂糖滴上,滴加液体培养基,25℃、光照16小时、光强4000lux下培养。3～5天细胞分裂;1周左右形成4个或更多细孢团;3～5周形成小愈伤组织。愈伤组织达2～3mm时,移到愈伤组织固体培养基上;直径达1cm时,移到固体分化培养基上,分化出根和新枝。

The following 8 F1 hybrid cultivars of broccoli were employed: Green Charger, Green Valient (these two cultivars came from Japan) 2Futura, Skiff, Naptune, Inwaprium, Coryet and Atlantic (these six cultivars came from Germany) . The leaves of propagated plantlets in vitro were cut into 1-2mm pieces with a wash solution(a) for plasmolysis, the'n were 'incubated in 10ml enzyme solution(b), and were placed on a rotary shaker(50 rpm) under light(2500 lux)at 21C for 3 hours. The mixture was filtered through a 45 um nylon screen (or stainless steel net) into centrifuge tubes and centri-fuged 5 min at 100 x g. The pellet was resuspended in 3.5ml 0,5M sucrose solu- tion(c) and 1.0 ml wash solution(a) was layered on the top and the tubes centrifuged atl00xg for 5 min.The floating grpen 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 x 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 3 cm plastic petri dishes. The agarose drops were solidified at once with a cold ironplate. 2-3 ml 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 x 20 x 7cm) with close Hd 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 25C 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 4-8 colonies.Most 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 6-8 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-solidified medium Girmen G (h). 5 months after isolation some roots and shoots from these calluses were regenerated.We have obtained cell division, celled colonies and p-calli in Green charger, Green Valient and Naptune, calluses in Skiff and Futura, roots and shoots regeneration in Futura.The solutions and media are listed as follows, (mg/1)(a)Wash solution. 100 MES + 100 CaCl2 + 100 NaH2PO4 +0.3 M mannitol, PH 5.5-5.8(b)Enzyme solution:2%cellulase + l%macerase in 10 ml solution(a) , pH6.8(c)Sucrose solution:0.5 M sucrose + 5 m M MES, pH5.5-5.8(d)WTl9/l medium, basic medium T* + 0.15M sucrose + 0.25M mannitol + 2,4-D 1 + NAA 1 + BAP0.5, pH 5.5-5,8, filter sterilized (e)WT29/l medium, basic medium T + 0.15M sucrose + 0.075M mannitol+ 2,4-D 1+NAA l+BAP 0.5, pH 5,5-5.3, filter sterilized(f)Girmcn 3 medium. Girmcn basic medium**+0.4M gluicose + 2,4-D 1+ NAA l + BAP 0.5, PH5.5-5.8, filter sterilized(g)Girraeri C medium, Girmen basic medium +34. 2g/L sucrose + 2 ,4 - D 0.1 + NAA 1 + BAPl, PH5.5-5.3, filter sterilized(h)Girmen G medium. Girmen basic medium + ICg/L sucrose + IAA0.1 + BAP 0.5 + 8g/L agar powder., pH5.5-5.8(i)Agarose. 1% agarose in solution (a)