Micropropagation Of Paeonia ’Bartzella’ -A Hybrid Between Herbaceous And Tree Peony

The cross between the Chinese herbaceous peony and the tree peony were called ’Itoh’ hybrid. More than hundreds of ’Itoh’ hybrid cultivars have been registered in the world, and have formed a peculiar variety group in Paeonia. However, ’Itoh’ hybrid had extremely low reproductive rate, which resulted in higher market prices, and then severely obstructed the industrial development. In recent years, the micropropagation technique of herbaceous and tree peony have been made a breakthrough. The plantlets with root have been obtained successfully in many researches, but seldom reported about ’Itoh’. This study firstly used its buds as explants, and in-depth studies of sterilization of explants, prevention and cure of browning, choosing of plant growth regulators and their concentrations and combinations, selection and improving of basal medium, rhizogenesis, and transplant of rooted vitroplants was carried out. The main results were as follows:(1) Sterilization of explants:buds of 8 intersectional hybrids between tree and herbaceous peonies in Paeonia was experimented under in-vitro culture condition, of which’Bartzella’,’Copper Kettle’ and ’Hillary’ showed better germination rate (≥90%) and multiplication rate (≥2.9) at initiation culture stage. ’Bartzella’was selected to develop a protocol for micropropagation infurther study, the results of which showed that the sub sprout tillers had the highest germination and multiplication rate; late March was the best sampling time and vitroplant with low contamination rate, high germination and proliferation rate (97.22% and 4.17%, respectively); the optimal subculture was two times because of the explants multiplication rate and the high level of differentiation capacity.(2) Multiplicaiton culture:’Bartzella’showed a serious browning in multiplication. In this study, we found that browning was lightened significantly by using medium with AgNO3 4.0 mg·L-1. Multiplication and growth of vitroplants were also improved by this method. In’Bartzella’, the optimal medium for shoot multiplication in in-vitro culture was WPM(double strength of CaCNO3)2)+ BA1.Omg·L-1+GA30.4mg·L-1+KT0.3mg·L-1+AgNO34.0mg·L-1.Modification of WPM basal medium on four-fold strength of Ca(NO3)2, keeping NO3-N concentration unchanged, reducing NH4NO3 concentration correspondingly were available to the improvement of multiplication and growth. For organic additives, chitosan had no significant effect on germination and multiplication rate of’Bartzella’. Instead, the hydrorysis of casein (0.25g·L-1) had significant effect. The best subculture cycle was 50d under the best multiplication culture.(3) Rooting culture:the rooting percentage was optimum when leaves were cut off completely. Shoots were cultured on 1/2MS for 10 days before the root induction-phase could help to balance hormone levels and improve rooting percentage. Browning in the test was lightened to some extent by using medium with activated carbon (AC), but has no significant effect on rooting. The optimal rooting protocol for’Bartzella’ was improved two-step method. Rooting percentage could be improved by a low temperature treatment (4℃) for 12 days under dark condition before the root induction-phase. It also prevented the callus inducement and improved the quality of rooting. Long root induction-phase time would reduce the quality of root and cause the necrosis of the base of root. The highest rooting rate and quality was obtained from each subculture of 30 days duration. The subculture times had a significantly influence on the rooting percentage of’Bartzella’. The rooting percentage was increased after 7 subcultures, but the quality was decreased.(4) Transplant acclimatization:30 days under 4℃ treatment could release the plantlets from dormancy. Then take the root number and callus size into consideration for transplant in later stage. After 60 days of transplant, the survival rate of the class 1 (root number> 3 and small callus) that had the highest survival rate, which was 73.58%. The plantlets were prone to die at the first month after transplanted. Growth of’Bartzella’ was improved when they were transplanted with arbuscular mycorrhizal fungi (AMF) of Glomus mosseae at 2.0g/plant.