Preliminary Studies On Plant Regeneration Via Somatic Embryogenesis And Agrobacterium-Mediated Transformation Of Camphor Tree (Cinnamomum Comphora L.)

Cinnamomum camphora L. is a large, handsome tree that widely cultivated in warm regions of the world as a specimen, street or shade tree; it has many valuable traits such as wide adaptability and resistance to environmental pollution. The wood is used for making cabinetwork, and the twigs, leaves and wood are used to produce camphor and camphor oil, which is an important export material of China. However, one mature tree can produce over 100,000 seeds a year, which are spread by fruit eating birds, and deep purple mature fruits from camphor tree begin to fall in October. A large quantity of fruits disperse widely around the tree, which contaminates not only the road surface but also passers-by' clothings under trees. The capability of camphor tree to bear numerous fruits restrains from exstension of this species as ornamental and shade tree to a certain degree. The species is also severely damaged by pests. There are two main pects of camphor tree: termites and moore. Termites destroyed many ancient and valuable camphor trees, it causes a tremendous economic lost, and the increasing occurrence of moore and termites has raised serious concerns about how to control these pests and protect camphor tree. To solve these problems, introducing desirable traits such as sterility and disease or insect resistance into this species is the mainly approach. However, Camphor tree is a heterozygous perennial tree with a long reproductive cycle that makes conventional sexual hybridization very time-consuming and low efficiency to improve tree characters. The application of genetic transformation is a promising strategy of camphor tree improvement.The major objective of this study was to breed camphor tree individuals with male sterility by introducing a chimera gene pTA29-Barnase that can induce male sterility in transformants through Agrobacterium tumefaciens-mediated transformation technology. For this purpose, an efficient in vitro propagation system and a regeneration system via somatic embryogenesis were developed firstly for a selected excellent source tree on the campus of Huazhong Agricultural University. Yellow, incompact granule-like embryogenic callus gradually appeared on white callus derived from cotyledon-like secondary somatic embryos after subcultures of 2 years. Embryogenic callus throughsuspension culture dispersed readily in liquid medium to give a very fine suspension of single cells and small aggregates. Subsequently, an efficient and reproducible protocol is developed for the regeneration of C. camphora protoplasts isolated from cultured embryogenic suspension cells. We also comprehensively studied and optimized parameters that affecting the efficiency of Agrobacterium tumefaciens'-mediated transformation of camphor tree by accessing gus transient expression, and a transformation protocol was established for the embryogenic callus. Finally, on the basis of the established transformation protocol, GFP gene and pTA29-Barnase were stably introduced into the embryogenic callus of C. camphora verified by PCR test. The major results of this study were introduced separately as following:1. An in vitro micropropagation system of C. comphora was developed from plumule of mature embryo. Callus was induced from leaflet explant of C. camphora. The results showed: The best bud proliferation was achieved on MS medium containing 1.0 mg/L BA and 0.1 mg/L NAA. The optimal auxins for the callus induction was obtained on MS medium supplemented with 0.1 mg/L CPA, 1.0 mg/L NAA or 1.0 mg/L 2,4-D; The optimal cytokinins for the callus induction was achieved on MS medium supplemented with 3.0 mg/L BA or 0.5 mg/L TDZ; Further development of callus into plantlets should be further studied. Shoots cultured for 6 weeks on proliferation medium differentiated adventitious roots 15-20 d after being transferred to rooting medium MS+1.0 mg/L IBA; the rooting frequency was above 50%.2. A plant regeneration system via Somatic embryogenesis was successfully established from immature embryos of C. camphora. Factors affecting somatic embryogenesis, proliferation of somatic embryos and plant conversion were investigated in details. The best carbohydrate source was sucrose; the best basal medium of embryogenesis induction was MS basal medium; 2,4-D has positive effect on somatic embryos induction of camphor tree. The highest frequency of induction was obtained in a medium containing 0.1 mg/L 2, 4-D and 1.0 mg/L BA in which 68.4% of zygotic embryos directly produced somatic embryos. Cotyledonary secondary somatic embryos were tested for conversion into plantlet after 6 weeks of interruption secondary embryogenesis on a medium containing 0.1 mg/L NAA, 3.0 mg/L TDZ and 0.5 mg/L GA3. The conversion frequency was up to 21.7%. The isolated shoots were subsequently incubated on MS medium with 0.1 mg/L NAA, 0.5 mg/L BA, and 0.5 mg/L GA3 for further growth, and rooted on MS medium with 1.0 mg/L IBA. Regenerated plants that transferred to soilhave normal appearance. Yellow, incompact embryogenic callus were gradually derived from somatic embryos after subcultures of 2 years and has been maintained for 3 years up to date. Embryogenic callus were transferred onto induction medium for somatic embryos initiation, maturation medium for somatic embryo maturing, and then to germination medium for shoot induction. Finally, the embryogenic callus could be induced to develop into plantlet. There is no difference in the conversion frequency between somatic embryos derived from embryigenic callus and cotyledonary secondary somatic embryos.3. Embryogenic callus of C. camphor a was choosed as incubation material for Agrobacterium tumefaciens-mtdi&Xtd transformation. Firstly, the sensitivity of callus to hygromycin B and kanamycin were tested in order to use the regeneration system to select and regenerate transgenic plants. Callus was shown to be highly sensitive to hygromycin B and when its concentration incressed to 50 mg/L in proliferation medium, the proliferation of callus was inhibited immediately. However, embryogenic callus showed much tolerance to kanamycin, and growth of 80% of the csllus was inhibited when the concentration of kanamycin reached to 100 mg/L. Conditions for efficient T-DNA transformation were comprehensively studied by assessing the transient expression of GUS gene. The results showed: the optimal bacterial density is OD^ = 0.6; the ideal time for embryogenic calli dipping in bacteria is 40 min and the optimal co-cultivation is 3 days. By infection according to this optimised T-DNA transformation protocol, and selection on selection medium supplemented with 50 mg/L hygromycin and 300 mg/L cefotaxime, we obtained stably transformed embryogenic callus according to PCR verification.4. Embryogenic callus containing GFP gene and a chimera gene pT429-Barnase were obtained. According to the established transformation system, and by using A. tumefaciens EHA105 containing NPTII gene and GFP gene; A. tumefaciens LBA4404 containing NPT II gene and a chimera vector (pTA29-Barnase), the transgenic callus of C. comphora were obtained. The results showed that the transformation protocol by introducing GUS gene to embryogenic callus of C. comphora could be widely applicable to A. tumefaciens-mediaied transformations, there is no significantly effect on tansformation of C. comphora between different selection gene and Agrobacterium strains with different genetic background; and PCR test showed stable integration of the foreign genes in embryogenic callus.5. An efficient and reproducible protocol is described for the regeneration of C. camphor a protoplasts isolated from cultured embryogenic suspension cells. Firstly, suspension culture system of embryogenic callus was established, the key point for very fine suspension cultured cells was to select new cells and subculture in time. Effects of incubation time, initial material and enzyme concentrations on protoplast isolation were investigated in details. The optimum incubation time is 12 h; the embryogenic suspension cells is the best initiation material for protoplast isolation; When they were decomposed for 12 h in an enzyme mixture solution containing 3% cellulase, 3% macerozyme, 0.12% MES, 0.011% NaH2PO4 ? 2H2O, 0.36% CaCI-2 * 2H2O, and 12.7% mannital, the optimum yield and viability of protoplast were obtained. Maximum protoplast yield (13.1±2.1xlO6/g FW) and viability (91.8±3.8%) were achieved using a mixture of 3% (w/v) Cellulase Onozuka RIO and 3% (w/v) Macerozyme Onozuka RIO in 12.7% (w/v) mannitol solution containing 0.12% (w/v) MES, 0.36% (w/v) CaCl2 ? 2H2O and 0.011% (w/v) NaH2PO4 ? 2H2O. First divisions occurred 7-10 days following culture initiation. The highest division frequency (24.6±2.9%) and plating efficiency (6.88±0.8%) were obtained in MS liquid medium supplemented with 30 g/L sucrose, 0.7 M glucose, 0.1 mg/LNAA, 1.0 mg/L BA and 1.0 mg/L GA3. After the proceeding of somatic embryo induction and shoot induction, the protoplast-derived calli produced plantlets via somatic embryogenesis at an efficiency of 17.5%. Somatic embryos developed into well-rooted plants on MS medium supplemented with 1.0 mg/L IBA. Regenerated plants that transferred to soil have normal morphology.