Establishment Of Regeneration System And Transformation Of Atractylodes Macrocephala Koidz

Atractylodes macrocephala Koidz is a perennial medicinal plant of compositae. Wild Atractylodes macrocephala resources decrease year after year, and most of the medicinal sources are cultivated varieties. Threatened by all kinds of diseases and insect pests in recent years during the period of cultivation, the quality and yield of Atractylodes macrocephala have been seriously affected. Transgenic technology is an effective means of genetic improvement. Introducing resistance genes by transgenic technology to enhance the disease resistance of Atractylodes macrocephala is worth exploring.This experiment adopted Atractylodes macrocephala cultivar, establishing an Atractylodes macrocephala in vitro regeneration system and a genetic transformation system of Atractylodes macrocephala mediated by Agrobacterium with high-performance. By means of introducing the inhibitor of apoptosis gene p35 and iap respectively from alfalfa twill armyworm insect baculovirus and beet armyworm into Atractylodes macrocephala koidz, the transgenic plants were obtained.The main results were as follows:1.The optimum culture condition of euphylla, stem apex, hypocotyl and epicotyl explants to induce polygerm was MS+TDZ 0.1 mg/L, 0.2 mg/L, 0.2 mg/L, 0.2 mg/L, and the differentiation rate could reach 47.5±3.96%, 75.1±4.41%, 78.7±7.11%, 88.4±5.34% respectively; the maximum propagation coefficient of the epicotyl, the best explant, could be 15.9±0.64. Taking advantage of 1/2MS+NAA 0.1 mg/L would promote the multi-buds rooting with the rooting rate of 90.3±0.43%.Transplanting survival rate could be 93.3%。2.Taking p Bar-Gus as a carrier, the Agrobacterium transformation system of Atractylodis macrocephala was built. The infection lasted 20 min under the condition of 0.4 bacteria concentration, but when adding 200μmol/LAS during the co-culture under 2d condition, the PCR detection showed that the conversion rate reached 15.8%; The tissue, polygerm, root and T1 floret with resistance after the transformation and screening were Gus staining positive. After T1 plant was detected by the test strip, the expression of bar gene was made, with a positive rate of 76%. The result of the spraying of 150mg/LBasta solution showed that the transgenic plants had significantly improved Basta resistance.3.The target gene p35 and iap were successfully induced into Atractylodes macrocephala. The conversion rate of T0 plants detected by PCR was 12.70%.; after carrying out Botrytis cinerea in-vivo inoculation of the plants detected by PCR and test strip as positive, the result showed: 9 of 25 Atractylodes macrocephala plants’ resistance to Botrytis cinerea was enhanced, creating the available breeding materials.4. 4 T0 p Bar-Gus plants were measured by fluorescence quantitative PCR and the copy number of bar gene turned out to be 2 to 3. The PCR test of T1 generation plants gained by propagation showed that the isolation ratio of the four strains was respectively 3:1, 3.67:1, 3.75:1 and 4.5:1,which indicated the possibility of just one copy number was relatively large. But the exact copy number was required to be confirmed by Southern blot.