Induction Of Petunia Callus And Isolation And Cultivation Of Petunia Protoplasts

Petunia（Petunia hybrida）is a perennial herb of Solanum pumila,native to South America,Argentina,rich in color,it can be widely used in indoor and outdoor potted plants and flower beds.It’s the world’s most widely used herbal flowers.However,due to the limitation of gene resources and the metabolic characteristics of anthocyanin,the yellow flowers and orange flower varieties of petunia are very rare.At present,petal breeding technology is adopted in petunia,and plant protoplast fusion technology can overcome the difficulties of poor compatibility.For the cultivation of new varieties of petunia to open up new ways.In this study,Petunia hybrida’Dream’was used as the experimental material for the induction of callus induction,protoplast isolation and purification of petiole hybrida cultivar SH-2015-1 The protoplast isolation and purification system of callus was established to lay a foundation for the further development of protoplast culture and cell fusion of petunia,and germplasm resources for the cultivation of new varieties of petunia.1.Callus induction of petuniaThe callus induction medium was cultured with MS+6-BA（1.0mg/L）+NAA（0.1 mg/L）,and the culture temperature was（24±1）℃,The callus was weighed at 14 h.d^-1,and the light intensity was 3000 lux and 6-7 d.The yellow-green callus was formed at the midrib and margin incision.The callus was cultured on the surface of the culture medium for 15 days.The callus induction rate up to 67.5%and was transferred from the primary culture medium into MS+6-BA（0.5 mg/L）+NAA（0.5 mg/L）medium for subculture,and the medium was replaced every 2 weeks.The organization provides nutrition and maintains undifferentiated state.2.Isolation and Culture of Protoplasts from Petunia（1）Establishment of Protoplast Separation SystemThe optimal combination of enzyme solution was 2%cellulase R-10+0.5%pectinase Y-23+0.3%segregation enzyme R-10,After centrifugation at 0.5 mol/L mannitol for 12 h in dark condition,the cells were centrifuged at 1100 rpm for 2 min at room temperature and then washed twice with the same rotation speed and centrifugation time to obtain purified protoplast yield and activity Respectively,to 3.4×106/g and 83%.（2）Establishment of protoplast culture systemIn this experiment,the osmotic pressure concentration,culture density,hormone combinations and other factors were compared,through a large number of repeated tests,no obvious protoplast cell division was observed,yet not suitable for the protoplast culture system.The experiment found that protoplasts ruptured 3 days after culture up to 50%protoplasts were dissolved and dispersed 1 week.Petunia callus protoplast culture system to be further studied.