Establishment And Optimization Of In Vitro Regeneration System Of Herbaceous Peony Based On Mature Embryo And Underground Bud

Paeonia lactiflora Pall.is a famous ornamental plant at home and abroad with great commercial value.However,tissue culture technology widely used in modem commercial flowers has not been successfully applied to plants of the genus Paeonia,which not only limits the large-scale production of herbaceous peony,but also makes research in molecular biology difficult.In this study,the optimization of the mature zygotic embryo and the underground bud in vitro culture of the herbaceous peony was provided,which provided technical support for the establishment of the rapid propagation system of herbaceous peony.At the same time,the tissue sectioning technique was applied to track the occurrence of adventitious roots and reveal the mechanism of occurrence,which provided a theoretical basis for solving the problem of rooting of herbaceous peony.In addition,this study explored the indirect induction pathway of somatic embryos and tried to solve the problem of callus re-differentiation by inducing the occurrence of embryogenic callus.The main results are as follows:1.WPM medium is the optimal basic medium for in vitro culture of zygotic embryo of 'Hang Bai',and 1/2MS(Ca2+ doubling)is the optimal basic medium for'Fen Yu Nu'.The optimal ratio of hormones in the initiation and proliferation stages was 6-BA 1.0 mg·L-1+GA3 0.5 mg·L-1.The number of subcultures of 'Hang Bai'tissue culture seedlings should be controlled within 4 times.The best rooting medium was 1/2 MS(Ca2+doubling)+IBA 1.0 mg·L-1+putrescine 0.5 mg·L-1.2.Among the five cut flower peony varieties,'Karl Rosenfield' has the lowest pollution rate and is most suitable for cluster bud induction.1/2 MS(Ca2+ doubling)+0.5 mg·L-1 6-BA+0.2 mg·L-1 KT is the best proliferation medium,and the growth coefficient of 'Karl Rosenfield' in this medium is 6.08,and substantially no browning and vitrification occur.At the rooting stage,the third-stage seedlings with a plant height of ? 3 cm and a large number of leaves had the best rooting effect,and the plant growth regulator had the best effect of adding 1/2MS medium with 1.0 mg·L-1 IB A alone.3.The occurrence of adventitious roots of peony requires the induction of plant growth regulators,which is an induced rooting type.The occurrence of multiple sites and unsynchronized roots is an important reason for the difficulty in rooting.After the inoculum cultured seedlings were inoculated into the rooting medium,the vascular bundles at the stem and the callus with strong division and differentiation will gradually form closely arranged and clustered root primordial cells.The root primordial cells divide and differentiate to form a spherical root primordium,and continue to grow to the epidermis,and finally break through the epidermis to form adventitious roots.4.The induction efficiency of embryogenic callus from high to low is:zygotic embryo>cotyledon>hypocotyl.The combination of NAA and TDZ is beneficial to the induction of embryogenic callus.When the hormone combination is NAA 0.5 mg·L-1+TDZ 1.0 mg·L-1 or NAA 0.5 mg·L-1 TDZ 0.5 mg·L-1,mature zygotic embryos of 'Hang Bai' can induce loose,yellow-green and granular embryogenic callus.The pre-culture time of cotyledons and hypocotyls should not be too long,and it should be within 40 days.The best hormone combination for cotyledon-induced callus was MS+2,4-D 2.0 mg·L-1+TDZ 0.2 mg·L-1+CH 300 mg·L-1;The best hormone combination for hypocotyl-induced callus was MS+ 2,4-D 2.0 mg·L-1+NAA 0.5 mg·L-1+ TDZ 0.2 mg·L-1.