Camphor Tissue Culture And Establishment Of Regeneration System And The Plant Expression Vector Pcambia2300. Aca

Cinnamomum camphora (L.) Presl. belonging to the Lauraceae family, is mainly cultivated in warm and humid areas of the world. This evergreen subtropical tree has been regarded as a useful wood for ornament, timber, medicine, and industrial raw materials. Conventional breeding method of C. camphora is propagating new plants from seeds. This method often leds to greatly genetic differentiation and reduces the valuable characteristics such as some stress resistance in the offspring. Therefore, the establishment of high efficiency and rapid propagation and regeneration systems of C. camphora using plant-tissue culture technology could maintain the fine breeding lines by genetic cloning, expand the scope for planting, obtain better economic and ecological benefits, as well as laid the groundwork for future genetic transformation.Amaranthus Caudatus Agglutinin (ACA) is a kind of storage protein existed in the seed of Amaranthus caudatus, belonged to amaranth lectin family. The plant lectin of ACA is insecticidal toxin to homopteran pests. If the ACA gene was transferred into the plant, the resistance to insects of plant would greatly improve. Therefore, in order to offer a suitable vector for genetic breeding, a plant expression vector with gene ACA (pCAMBIA2300-ACA) was constructed.The results of this dissertation were as follows:1. The method optimization of tissue culture of seeds in C. camphora. The best disinfecting method for the shelled seeds of C. camphora:explants were dipped in 75% ethyl alcohol for 45s, then agitated in a solution of 0.1% HgCl2 for 5min, and rinsed with sterile water for 5 times lastly. MS medium is the best medium for seed germination, in which the germination rate was 99%, and shoots were strong.2. The rapid propagation system was established. The optimal medium for introduing multiple shoots was:MS +6-BA4.0 mg/L+NAA0.5 mg/L; The best medium for buds proliferation was:MS +6- BA2.0 mg/L+NAA0.5 mg/L; Al 1 of four-linked small shoot s could form crowded shoots and their propagation coefficient could reach 8.5.3. The regeneration system was established. The upper leaf was the most suitable explant for regeneration. The best medium for callus induction and adventitious buds differentiation was:MS +6-BA5.0mg/L+NAA1.0 mg/L; Rooting medium of 1/2 MS+IBA1.0 mg/L+NAA0.5 mg/L exhibited the highest rooting rate of 93%. A survival rate of 85% can be achieved for regenerated plantlets after acclimatization and transplantation.4. Constructed the plant expression vector (pCAMBIA2300-ACA). The gene ACA was subcloned from pGEM-ACA to construct the expression vector pCAMBIA2300-ACA. The vector contained "CaMV 35S promotor-ACA gene-OCS terminator sequence" and a NPTâ…¡gene promoted by CaMV 35S promoter. The sequence of the vector was verified by enzyme digesting and sequencing.