Somatic Embryogenesis And Callus Re-differentiation Of Herbaceous Peony

Paeonia lactiflora Pall.is a traditional flower in Chinese,improving breeding efficiency is basic requirements for its commercial and industrial production.There have not been a mature regeneration system in the peony tissue culture which restricts further molecular studies.In this study,plant regeneration of herbaceous peony was carried out not only by direct or indirect somatic embryogenesis but also by callus re-differentiation pathway.Through callus induction of flower organ,suitable organ type was screened to expand material range.The systematic study of callus browning and rejuvenation was carried out based on the above callus to ensure the long-term effective use of callus.The main resuhs were as follows1.Indirect somatic embroy genes is:Embryogenic callus generated after transferred the callus to medium with 2.0 mg/L 2,4?D in a 90 days' constant darkness environment.Globular embryos and heart embryos were generated after transferred the embryogenic callus to 1/2MS(Ca2+doubled)+6-BA 1.0 mg/L+NAA 0.2 mg/L medium for a constantly illumination cultivation of 60 days.There are exogenous and endogenous origins of somatic system.Exogenous includes origins from individual surface cells and origins from multiple subcritical cells.The splitting of embryogenic cells was symmetrical and the presence of asymmetric splitting cells was not found2.Direct somatic embryogenesis:Different hormone combinations of 6-BA,2,4-D,NAA,TDZ,KT can not promote direct somatic embryogenesis of zygotic embryos from herbaceous peony.Culture of zygotic embryos showed that 1/2MS(Ca2+doubled)+6-BA+GA3 promoted new leaves extraction from cotyledons.Combination of IBA and IAA promoted a large number of adventitious roots of herbaceous peony.Seedlings of hybridl(self-fertile,unnamed)were much more stronger than'Fen Yu Nu'.3.Callus re-differentiation study:The non-stretched stem segments of underground buds were ideal materials for callus re-differentiation.1.0mg/L or 1.5mg/L ABA combined with low concentration of NAA and 6-BA promoted callus transforming into pre-differentiation status.White and green bar-shaped adventitious buds tips generated after transferred the callus into medium with timely reduction hormone concentration.Hormone-free medium promote the growth of white buds tips into significantly visible gas root and gaps generated at the top of green buds tips which finally grew into two small leaves.The stem and leaf differentiation of the seedlings was obvious after transferred buds into medium with low concentration of 6-BA and 2,4-D.Roots could not be promoted with commonly used formula of IBA and IAA.4.Callus induction study of flower organs:Floral organ types were sorted in view of callus induction ability ovary>ovule>Petal>anther>filament.Petal base was more favorable than other parts for callus induction.Callus of ovary and ovule are indeed derived from the placenta tissue.Stamens and filaments directly died and the callus came from the mitosis pf pollen.1.0mg/L NAA and 1.0mg/L 2,4-D was suitable for all organ types producing callus.Using 150 mg/L VC alone was beneficial in relieving callus browning.1/2MS(Ca2+ doubled)medium with low concentrations of 2,4-D,6-BA,TDZ was suitable for callus rejuvenation.