Studies On Heterosis Breeding And Agrobacterium-mediated Genetic Transformation Of Chinese Pink

Chinese pink,which is usually used as annual or biennial. Because of its good characters, such as colourful flowers and long florescence,it is more and more accepted by the public. This study is kind of preliminary exploration for variety improvement of Pink, which makes use of the technology of Heterosis breeding and regeneration and genetic transformation.The experimental results are as follows:1. Heterosis breeding,In this study, first of all,purifit all of the inbred lines,and get 22 inbred lines.Second, selction of self-cross Pedigree and breeding of individual plant was debeloped to select 12 different ornamental traits.7 inbred lines of Chinese pink were selected as parents for crossing according to GriffingⅡ,7 selfcrossed combinations and 21 cross combinations obtained. Firstly, seed germination ratio of all the crosses(short for cross combinations and selfcross combinations) were compared; and seed germination ratio of different ripeness was discussed; secondly, the heterosis of main ornamental characters and inheritance laws of the ornamental characters of all the Dianthus chinese crosses were analyzed. The main results of this study were as following(1)Parent NO.1b had the highest average number of single-fruit seed is70, and NO.5 had the lowest average number of single-fruit seed is 36,21 hybrid combinations in the number between them; Significant heterosis in germination ratio was observed in Cross combinations seed. (2)According to the results of heterosis analysis, the Dianthus chinensis crosses had significant heterosis in the ornamental characters,while in the flowers density initiatorye,height the crosses showed transgressive heterosis. The flower single showed mid-parent heterosis, while the time of flower showed minus transgressive heterosis. (3) According to the results of heterosis analysis, combination analysis and heredity analysis of flower colors, ten excellent cross combinations were obtained, they were:H×31、H×41、H×LHD、LHD×31、LHD×41、5×31、5×H、Z×31、Z×H、31×41.2. The callus induction and plant regeneration from leaf explant of inbred line NO.1 in laboratory were studied.Results indicated that MS+2,4-D1.0mg/L+NAA 0.3mg/Lwas a benefical medium for callus induction of scratched leaves,the efficiency reached 90-95%;when the medium was supplemented with 1.0 mg/LNAA+1.0 mg/LBA,there was the highest proliferation efficiency;the callus grew better when the concentration of 2,4-D and sugar were 1.0mg/L and 60g/L respectively.The best medium for callus differentiation was MS+BA2.0mg/L+NAA0.1mg/L+activated carbon1.0g/L,and the adventitious buds grew well.Research about callus genetic transformation were conducted by studying the transformation factors influencing.In this protocol,the callus were infected by Agrobacterium tumefaciens for 10-13 minnutes without pre-culture,after 4d for co-culture the callus was tranferred into differentiation medium supplemented with 400 mg/Lcef and 50mg/LKm for selective culture.PCR analysis of the transgenic plants showed the positive rate was 36.4%. 3.The establishment of regeneration system and gene transformation for Chinese pink.Providing three materials(20,31 and40) of aseptic seedlings of Chinese pink from stem section. Research on the influence of the way take off the leaves between tearing and cutting,and different combinations of NAA and BA on shooting, turn out that tearing leaves from the stem is better, the optimal shooting medium for 20 line and 40 line is MS+BA1.0mg/L+NAA0.3mg/L;and MS+BA1.0mg/L+NAA0.5mg/L for 31(56.9%,93.2%,66.6%). By exploring the influence of different auxins on rooting, the research compares the effect of MS medium containing 0.1 mg/L NAA, IAA, IBA, and 1/2 MS medium. The result shows that the optimal medium for in-vitro rooting is MS+0.1 mg/L IBA. The aseptic seedlings in this medium has the highest rooting rate(100%), with most(5.17roots) and longest(3.69cm) roots. In transplanting the aseptic seedlings into soil, peat, peat+perlite, the result shows that in the medium of peat+perlite, it has the highest survival rate(75.3%).The establishment of transgenetic system of Chinese pink.This research employs the method of Agrobacterium tumefaciens-mediated genetic transformation system. The research shows that:Chinese pink is very sensitive to hygromycin, the selection pressure for leaf is 4mg/l in 20 line, and 5mg/l in 31 line and 40 line. By analyzing the transient expression of GUS, the research explores the effect of pre-culture time,inoculation time,co-culture time and the concentration of AS.The optimal transgenetic system is:pre-culture the leafs on regeneration medium MS+BA1.0mg/L+NAA0.3mg/L for2 days; inoculate for 10 minutes; and then co-cultivate for 4 days on medium of MS+BA1.0mg/L+NAA0.3mg/L+AS100mg/L; afterwards,transfer the leaves into selection mediumMS+BA1.0mg/L+NAA0.3mg/L+ Hyg5mg/L+Cef400mg/L.Change the medium for every 3 weeks. After transferring the target gene, the transgenetic rate of 20 line is 4.44%, and for the 31 line is 20.2%, and for parent 40 line is23.5%. After transferred the transgenetic plantlet into the soil, we studied the vase life and the potted flowering stage of 20 line and 40 line in bloom stage. The results demonstrated that,vase life of 4 antisense direct transgenic lines of 20 line was all prolonged by about 5 days; Among 12 direct transgenic lines of 40 line,5 lines vase life of was prolonged by about 3-4days.Potted single initiatorye of 4 antisense direct transgenic lines of 20 line was all prolonged by about 3-5 days; the group florescence of 20 line prolonged by about 14-20 days.5 lines of Potted single initiatorye of 12 direct transgenic lines of 40 line was prolonged by about 4-6 days; the group florescence of 40 line prolonged by about 9-15 days.