Study On In Vitro Regeneration Via Meristematic Nodule Culture And Embryo Culture In Tree Peony

The research and application of tissue culture and the establishment of a modern breeding and propagation technology system have gradually become an inevitable requirement to promote the industrialization of tree peony.Plant regeneration via meristematic nodule（MN）and embryo culture are two important tissue culture techniques,which have broad application prospects in the expansion of elite clones and breeding practice.Thus,this research broke through the bottleneck of MN differentiation and established a protocol of in vitro regeneration system with MN culture,through explant selection,medium selection and hormone adjustment,combined with histological,cytological and physiological observation.Meanwhile,key factors affecting in vitro embryo culture were explored,and the seedling method with embryo culture and micropropagation were established.This study will be beneficial to the breeding and propagation of tree peony.Below are the main results:1.A protocol of in vitro regeneration via MN culture was established firstly in P.ostii‘Feng Dan’with cotyledons as explants,including five consecutive steps of embryogenic callus（EC）formation,MN induction and leaf cluster differentiation,shoot elongation,rooting and acclimatization.Upward adaxial surface was the best inoculation method for callus induction of cotyledons after 15 days of culture,and the highest EC induction rate（87.50%）was achieved when cotyledons were cultured on modified murashige and Skoog（m MS）medium with 1.0mg/L N-（2-chloro-4-pyridyl）-N-phenylurea（CPPU）+1.0mg/Lα-naphthylacetic acid（NAA）for 30 days.The optimal MN induction rate（100%）and leaf cluster differentiation rate（45.83%）were obtained when ECs were cultured on modified woody plant medium（m WPM）supplemented with 0.5mg/L CPPU+0.5mg/L thidiazuron（TDZ）for 12 times with a subculture time of 10 days.The combination of 0.25mg/L 6-Benzyladenine（BA）+0.2mg/L gibberellic acid（GA₃）yielded the best shoot elongation（13.40 shoots per nodule）,rooting rate（43.33%）and consequently survival rate（45.83%）.Histological,cytological and physiological studies showed that the decrease of Indole acetic acid（IAA）/cytokinin ratio and the increase of putrescine（Put）,spermidine（Spd）and spermine（Spm）contents were important factors for the induction and differentiation of MN.2.A protocol of embryo culture was achieved in P.ostii‘Feng Dan’and P.rockii‘Jing Hong’with mature embryos at 90 days after anthesis（DAA）as explants,and it has been successfully applied in the embryo culture of distant hybrids between tree peony and herbaceous peony for the first time.Combination of activated carbon（AC）and GA₃was the key to seedling establishment,and the best seedling establishment medium was MS+0.6g/L AC+0.5mg/L GA₃+0.5mg/L IAA;The type I seedlings after 50 days of culture was the best choice for acclimatization,and this method can shorten the cycle of seedling establishment to 50 days;With this method,the seedling rate of cracked seeds with traditional method of sowing can be increased from 0.61%、10.20%、4.65%to 1.47%、17.95%、22.50%in‘Martha W.’בGolden Era’,‘Martha W.’×07-LB-1 and‘Martha W.’בBlack Pirate’,respectively.3.The inoculation method and embryo age were key factors for immature embryo culture in P.ostii‘Feng Dan’.Upward micropyle with placenta was the best inoculation method for initiation culture of immature seeds,and this method advanced the successful in vitro immature embryo culture to 30 DAA（cellularization stage of endosperm）,indicating the endosperm cellularization stage played an important role for immature embryo culture.The effect of immature embryo culture and micropropagation was improved with embryo maturity.Meanwhile,a protocol of micropropagation was established in P.ostii‘Feng Dan’with mature embryos（90 DAA）as explants.The best embryo germination medium was m MS medium+0.5mg/L BA+1.0mg/L GA₃;The best medium for shoots proliferation was m WPM+0.25mg/L BA+0.2mg/L GA₃+0.1mg/L Kinetin（KT）,with the highest number of shoots（8.10）;The best root induction medium was 1/2MS+1.5mg/L Indole-3-butyric acid（IBA）+1.0mg/L Put+30mg/L phloroglucinol with the highest rooting rate（41.48%）and survival rate（33.92%）.4.The best treatment for transplantation was obtained with seedlings of‘Jing Hong’as explants,by studying the effects of AMF and exogenous Put on the growth of seedlings.The results show that AMF inoculation and exogenous Put can significantly improve the survival rate and parameters of plant growth,and overcome the problems of slow growth and decreased survival rate after seedlings were transplanted for a period of time.The promotion effect of dual application of C.E inoculation and exogenous Put was better than sole treatment,with 0.05mmol/L Put in the C.E.-inoculated treatments being the most effective.Physiological studies showed that the ratio of endogenous Spd,PAs and Spd+Spm/Put in roots increased significantly,and the ratio of Put/PAs decreased significantly,which may be the important factors for the improvement of parameters with AMF inoculation and exogenous Put treatment.This technology improves the effect of embryo culture,and also provides a reference for the transplantation in other regeneration systems.