| Chicory (Cichorium intybus L.) is a member of the compositae family and a perennial herb, widely distributed in the world. It is one of the major forage and vegetable widely utilized at home and abroad. C. intybus L.contains large numbers of pharmaceutically important phytochemicals such as sesquiterpene lactones, glycosides, flavonoids, coumarins, anthocyanins, organic acids,and cytokinins. C. intybus has rich nutrition, but protein content is very few in its body. Phytoprotein is main source of protein for human and haplo-stomach animal, but the plant nutrition is not overall such as low content lysine and tryptophane in seed protein of grain crop and lack of methionine and cysteine in pulse and greengrocery. Sulphur-containing amino acid (SAAs) plays an imimportant role in improving the milk and meat quality and wool yield and the weight of livestock. It can increase the content of SAAs in C. intybus L.and improve the quality by utilizing the genetic transformation technology.Based on the construction of high frequency regeneration system and genetic transformation system of chicory (C.intybus L.).γ-zein and zeolin gene were transformed into cnicory leaf disc via Agrobacterium infection transformation. The transformed plants were gained. Molecule detection and expression location were carried out. It was showed that the exogenous gene had integrated into the genome of chicory. Now the major results studied in the paper are showed as followings:1. Effect of basal medium, genotype, explants, hormone type and concentration on regeneration of chicory were studied and a reliable and efficient regeneration system was established. The optimal regeneration medium for the euphylla of C.intybus L.cv.Puna was MS+6-2.0 mg/L BA +0.2mg/L IBA, with the max callus induction frequcy being 98.92% and the regeneration rate being 93.74%, the mean number of shoots per explan being 19.46.The regenerated shoots were transferred to 1/2 MS medium supplemented with 0.1mg/L NAA for rooting, and it was very easy to root and the rooting rate was 98.77% and survival rate was over 95%.2. The genetic stability of regenerated plants was analyzed using RAPD markers. The results of RAPD amplification showed that the genetic stability of the regenerated plantlets with the first culturing was maintained, though slight variations were found with the second-culturing regeneration plant. This suggested that in vitro regeneration were a good rapid propagation way to obtain genetic stable descends of chicory.3.Introduced by the cloning vector pMD18-T,γ-zein and zeolin gene and plasmid pCAMBIA1302 were joined, thus plant expression vector pCAM-γ-zein and pCAM-zeolin with selective marker hpt gene were constructed. The gene was controlled by CaMV35S promoter. The recombinant plasmid was transformed into Agrobacterium tumefaciens LBA4404 for genetic transformation.4.Establishment of a genetic transformation selection system for chicory:Transformant was cultured on pre-medium with no hyg for 1-2d after the co-culture, then add 25mg/L hyg at callus cultivation and shoot regeneration stage, while 15mg/L hyg at enlarge propagation period and 10 mg/L hyg at rooting period. Cefotaxime (Cef) was used for the bacteriostasis in Agrobacterium infection transformation and it was 500mg/L at callus cultivation and regeneration stage while 250mg/L Cef at rooting stage.5.Optimized protocols of Agrobacterium-mediated transformation for chicory and plant regeneration were developed by orthogonal design.The results indicated that the following conditions were outstanding for the improvement of transformation efficiency of chicory:Addition of 2mg/L6-BA and 0.2mg/LIBA to leaf Pre-culture medium, Pre-culture time of 2d, bacterial suspension OD600 of 0.4Abs, infection time of 10min, co-culture medium pH of 5.4-5.6, co-culture time of 3d and supplementation of 100umol/L acetosyringone to Agrobacterium co-culture medium.On the basis of the above studies, Agrobacterium-mediated transformation system of chicory euphyll has been set up. For verification the Agrobacterium-mediated transformation system,γ-zein gene and zeolin gene were incorporated into genomes of chicory, the result showed that the average resistant frequency of hyg was 13.52%.and it was 3.58 times more than that of basic transformation condition.6. Resistant plants were detected by PCR assay. PCR-Southern,Dot blot,Southern hybridization, RT-PCR detection and analysis identification of hygromycin resistance of in vitro leaves, and the result showed that the exogenous gene had integrated into the genome of chicory and were transcribed on RNA level.7.γ-zein gene was expressed in cell nucleus of leaf epidermis cell and cell membrane of root tip of transgenic chicory by confocal microscopy localization8. 14 positive plants withγ-zein gene and 12 positive plant with zeolin were obtained by this experiment,the positive plant frequency of transformationγ-zein gene and zeolin gene was 42.42% and 46.15% based on numbers of PCR, respectively. The averaged transformation frequency of two genes was 5.6% and 6.38%, respectively, based on numbers of resistant frequency and PCR of regenerated transgenic plants.
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