Protoplast Isolation Culture And Plant Regenration Of Petunia And Calibrachoa

Petunia(petunia hybrida)is praised as the king of flower plants.Atpresent,although pentunias are rich in colour,the anthocyane metabolism characteristics and the limitation of genetic resource,yellow and orange flower species are extremely rare.Calibrachoa and petunia are of the same family,but not different genera.Calibrachoa is becoming an important new horticultural plant and has many yellow and orange varieties.Calibrachoa can provide gene resources for cultivating yellow varieties of petunia.But the relationship is relatively far,there is an obvious sexual hybridization barrier and it is difficult to get filial generation.Plant somatic cell fusion technology can overcome the difficulties of poor cross incompatibilities and provide a way for breeding new germplasm resources.Use Petunia hybrida'Shock Wave' and Calibrachoa hybrid 'lindura yellow' as materials,we studied that the factors affecting the mesophyll protoplasts isolation culture and regenrarion,we establish appropriate system for the varieties of protoplast isolation and culture,to lay a foundation for breeding new varieties.1.Petunia protoplast isolation culture and regeneration(1)The establishment of the protoplast isolation systemWe investigate the key factors such as mannitol concentration?enzyme?time and centrifugal acceleration that affecting isolation efficiency of mesophyll protoplasts.The results show that,it is the sutiable isolation conditions that the optimal enzyme solution is consisted of 2%Cellulase R-10+0.2%Pectolyase Y-23+0.4%Macerozyme R-10+20 mM MES+0.1%BSA+0.11%CaCl2,incubated the leaves at 0.5 M mannitol with no shaking for 5 h.1100 rpm centrifugal 2 min for precipitating protoplasts,putting acceleration and brake settings of the centrifuge to zero.Protoplast yields were up to 2.9×106 protoplasts per gram and the viability was 88.1%.(2)The protoplast culture and regenerationWe investigate the key factors such as culture system?hormone concentration and culture system that affecting culture of mesophyll protoplasts.The results show that,it is the sutiable culture conditions that purified protoplasts were cultivated on the solid medium of KM8P+0.5 mg/1 6-BA+2.0 mg/1 NAA at a density of 0.5×105 protoplasts ml-1.After 24? constant temperature culturing in the dark condition,small cream calluses were visibled by eye in 17 days.One month later,microcallus were transferred to the proliferation medium of MS+2%sucrose+0.7%agar+0.5 mg/1 6-BA+0.5 mg/1 NAA,temperature was(24±1)?,light time was 14 hours per day and illumination was 3000 lux.20 days later,5 mm of microcallus were transferred to the differentiation medium:(1)MS+2%sucrose+0.7%agar+0.5 mg/1 6-BA+0.1 mg/1 NAA,roots was differentiated after 13 days.(2)MS+2%sucrose+0.7%agar+1.0 mg/1 ZT+0.1 mg/1 NAA,buds was differentiated after 16 days.2.Calibrachoa protoplast isolation and culture(1)The establishment of the protoplast isolation systemWe investigate the key factors such as mannitol concentration?enzyme?time and pretreatment method that affecting isolation efficiency of mesophyll protoplasts.The results show that,it is the sutiable isolation conditions that Under the dark pretreatment 24 h at 4?,then young leaves were incubated 5 hours in the optimal enzyme solution of 2%Cellulase R-10+0.2%Pectolyase Y-23+0.8%Macerozyme R-10+0.25 M mannitol+20 mM MES+0.1%BSA+0.11%CaCl2.Setting the acceleration and brake of the centrifuge to zero,1200 rpm centrifugal 2 min,protoplasts yield and viability was 5.60×106 per gram and 870%.(2)The establishment of the protoplast culture systemWe investigate the key factors such as culture system?hormone concentration and culture system that affecting culture of mesophyll protoplasts.The results show that,it is the sutiable culture conditions that purified protoplasts were 24? constant cultivated on the solid medium of KM8P+0.5 mg/1 6-BA+0.5 mg/1 NAA,at a density of 0.5 to 1.0×105protoplasts ml-1 in the dark condition.3 days later,protoplasts regenerated the cell wall,and begun to divide at the rat of 0.6%after 4 or 5 days.3 weeks later,white small calluses were visibled by eye on the medium.