The Expression Of Chimonanthus Praecox Cplectin Gene In Lily And The Analysis Of Resistance To Insect

In this paper,the high-frequency regeneration and the heredity transformation system of Lily were constructed by regenerating the explants,optimizing the antibiotic screening and the Agrobacterium-mediated genetic transformation condition with Lilium longiflorum "White-Elegance" and "Gelria" as tested materials.The Cplectin gene has been successfully recombined into the genomes of lily and the anti-insect experiment was carried on in the transgenic lines.The main results were as following:1.Establish the high-frequency regeneration system(1)The results showed that the most suitable medium for inducing the scales of "White-Elegance" to differentiate adventitious buds was MS+6-BA 0.5 mg/L + NAA 0.2 mg/L,the inductivity rate is 91.7%;and the most suitable medium for the scales of the regenerative plants to differentiate adventitious buds was MS+6-BA 1.0 mg/L + NAA 0.2mg/L,the inductivity rate is 90%; the most suitable medium for the petiole of the regenerative plants to differentiate adventitious buds was MS+NAA 1.0 mg/L,the rate is 77.5%.And the rooting medium is 1/2 MS + NAA0.05 mg/L.(2)The results showed that the most suitable medium for inducing the scales of Lilium longiflorum "Gelria" to differentiate adventitious buds was MS+6-BA 0.5 mg/L+NAA0.05mg/L,the inductivity rate was 88.3%;the best medium for regeneration from callus was MS+6-BA 1.0 mg/L +NAA 0.1 mg/L+ sugar 30g/L+ agar 6g/L,the inductivity rate is 250%;the medium for keeping the embryogenic callus was MS+6-BA 0.5mg/L+NAA 0.05mg/L+ sugar 30g/L+agar 6g/L.And the most suitable medium for the scales of the regenerative plants to differentiate adventitious buds was MS+ 6-BA 1.0 mg/L + NAA 0.1 mg/L,the inductivity rate was 87.5%.And the rooting medium is 1/2 MS +NAA0.05 mg/L.2.The establishment of genetic transformation system (1)The selection of receptor materialFor "White-Elegance",the inductivity rate of differention for the scales and petiole of the regenreative plants was both high.The scales organization has a strong ability for differention and to resist Agrobacterium,so there would be more resistance buds,but the false positive transformation receptor rate was relatively high.Consequently,it chould increase the workload of later detection. However,the petiole also has strong renewable capacity,and the most of them can regenerate plants.it also can reduce the rate of variation yet,so we choose "White-Elegance" petiole of the regenerative plants as the receptor material.For "Gelria",there were a lot of loose yellow embryonic calluin in the process of regeneration, and strong vitality could be kept after repeatedly subculture.If put them into the suitable medium for inducing adventitious buds,they could grow out renewable small plants.It was very favorable in genetic transformation,so we choose "Gelria" embryonic callus as the receptor material.(2)Determining the antibiotics concentrationAccording to kanamycin(Kan)resistance test,a feasible filtering concentration of receptor materials was determined.Using the "White-Elegance"petiole as the receptor materials,the rate of brown of the petiole was 97.5%with Kan 100 mg/L concentration which was the sublethal concentration,so Kan 100mg/L is suitable as the petiole resistance selection pressure.And also,using "Gelria"callus as the receptor,the rate of brown of the callus was 95.0%with Kan 75mg/L concentration which was the sublethal concentration,so Kan 75mg/L is suitable as the petiole resistance selection pressure.In the rooting culture,when Kan concentration was 150 mg/L,the leaves of the two types of regenerative plants were all yellowing,and there were no roots generation, so we choose Kan 150mg/L as a selection pressure for roots of plants.According to Cefotamine(Cef)antimicrobial tests,the receptor materials had good antibacterial effects and would not affect the growth of receptor when the concentration of Cef was 250 mg/L, so we choose Cef 250 mg/L as the inhibiting concentration.(3)Study on conditions of genetic transformationIn the test,several major factors of impacting Wansformation efficiency of Agrobacterium have been studied and results shows that:when using "White-Elegance" petiole as a receptor material, pre-culture 2ds,then put the petiole into the Agrobacterium bacilli with the MS(no NH₄NO₃) re-suspension for 10-12 mins with the OD₆₀₀of 0.6-0.8 and co-culture 3ds,higher instantaneous rate of 41.6%could be got;when using "Gelria" callus as a receptor,after 2-3 min deal with the trauma, put them into the Agrobacterium bacilli with the MS(no NH₄NO₃)re-suspension for 8-10 mins at the OD₆₀₀of 0.6-0.8,then co-culture for 3ds,higher instantaneous rate can be reached to 43.3%.Further study suggested the instantaneous rate of the two receptor is higher when we added 100 μmol/L AS into centrifugal re-suspended in the liquid MS(no NH₄NO₃)and put AS of 100μmol/L in the co-culture medium,and the rate is increased from 41.6%and 43.3%to 71.6%and 75.0% respectively.3.Obtain of the transgenic plants21 "White-Elegance"and 25 "Gelria" resistant plants were identified by kanamycin resistance screening,GUS histochemical staining and PCR ananysis to 86 "White-Elegance"and 93 "Gelria". All the analysis suggested that Cplectin gene has been successfully recombined into the genomes of the two material and the conversion rates respectively are 24.4%and 26.8%.4.Anti-insect experimentFor the anti-aphid tests,the results showed that:the two kinds of transgenic plants has higher insect-resistant than comparision after planting the positive plants...