Tentative Exploration Of Regeneration System And Differential Display Of The Different Developmental Flowers By CDNA-SRAP In Chimonanthus Praecox L.

Wintersweet [Chimonanthus praecox (L.) Link] is a deciduous shrub belongs to the Calycanthaceae family. It is a famous traditional fragrant flower shrub and landscape plant in winter. In recent decades, a lot of researches were developed in the germplasm resources, cultivar classification and garden applications of Ch. praecox, but as a woody plant, the reports about tissue culture are seldom, especially about regeneration to Ch. praecox. As the development of molecular biology, some related gene about stress resistance and floral scent were cloned, molecular study on Ch. praecox has become hot research field.In this study, plant regeneration system of Ch. praecox tentative developed by the TCL, embryogenesis and other methods.And differential display was studied on the different developmental flowers which from’H29’Ch. praecox tree by cDNA Sequence related amplified polymorphism(cDNA-SRAP).The main results were as follows:1. Tentative Exploration of plant regeneration system of Ch. praecox(1)Experiment with the hypocotyls and cotyledons of Ch. praecox as explants to the organogenesis regeneration.1/2MS basal medium with 0.6%(w/v) agar and 3%(w/v) sucrose, supplemented with 0.5 mg/L KT, and 0.2 mg/L NAA were optimal for callus induction and rhizogenesis. (2)Experiment with the hypocotyls and cotyledons of Ch. praecox as explants to indirect embryogenesis of regeneration. Loose callus with high vigour was induced from both two kinds of explants on 1/2MS basal medium with 0.6%(w/v) agar and 3%(w/v) sucrose, supplemented with 2.0 mg/L 2,4-D in dark, and callus induction percentage is up to 100%. The hypocotyls showed great ability to induce callus, followed by cotyledons.(3) Shoots showed weak, red leaves, internode shortening and so on after several times subculture on the 1/2 MS medium with 0.6%(w/v) agar and 3%(w/v) sucrose, supplemented with 1.0 mg/L 6-BA and 0.1 mg/L 1BA. On optimized MS medium with 0.6%(w/v) agar and 3%(w/v) sucrose, supplemented with 1.0 mg/L 6-BA,0.1 mg/L IBA and 0.2mg/LNAA. Shoots were vigorous, less red leaves and internode shortening, proliferation coefficiency was improved.2. Differential display on the different developmental flowers of Ch. praecox by cDNA-SRAPMolecular marker of cDNA-SRAP has many advantages, such as easy operation, low cost, good repetition and abundance of fragments. It is a valid method to the study on the differential display. PCR reactions were carried out with cDNA of flower buds and flowers which collected from august to December, and 19 pairs primers were selected from 170 pairs SRAP primers.82 fragments were cloned from 586 gene differential expression fragments obtained, then got 49 positive clones. After sequencing,23 fragments biological information was obtained by NCBI blast, including homology proteins of 5 fragments, some transcription factors, transposons, retrotransposons related sequence. Finally, we predicted the functions of these fragments.