| Atalantia buxifolia(Poir.)Oliv is an evergreen shrub or small tree belonging to the citrus subfamily of Rutaceae family.A.buxifolia can be used as a dwarfized root stock for garden plants,medicinal herbs and citrus cultivation,and used an important genetic resource for breeding of cultivated citrus.At present,the genome sequence of A.buxifolia has been sequenced,but its regeneration and transformation systems have not yet been established.In order to realize exploitation and utilization of wild resources and carry out genetic engineering breeding in the future,it is very necessary to establish a regeneration system.In this study,the stem segments and thin-layer cells in stem segments of A.buxifolia were used as explants to establish a tissue culture regeneration system of A.buxifolia through the organogenetic pathway.In addition,the immature ovules of A.buxifolia was used as explants,a regeneration system was initially established through the somatic embryonic development pathway.On this basis,a preliminary exploration of the genetic transformation mediated by Agrobacterium tumefaciens was carried out.The main research results are listed below:(1)Establishment of tissue culture and regeneration system of A.buxifolia through organogenesis pathway.The best disinfection method for A.buxifolia seeds was treated with 2% sodium hypochlorite for 10 min.The most suitable culture medium for induction of adventitious buds in A.buxifolia stem segments was MT + 0.5 mg/L 6-BA,with the induction rate of adventitious buds of 66.67%,and 3.7 buds per explants.The stem segments of A.buxifolia with a seedling age of 15 d were the most suitable for induction of adventitious buds,with the induction rate of adventitious buds of 72.92%,and the average number of buds was 5.36 per explant.The best culture method was dark culture for 30 d,and the induction rate of adventitious buds was the highest(89.73%).6-Benzylaminopurine(6-BA)was suitable to the induction of adventitious buds from the cells in the thin layers of A.buxifolia stems.When the concentration of6-BA was 0.50 mg/L,the average number of bud primordia was 5.26 per explant.Thidiazuron(TDZ)was suitable to callus induction from thin layer cells of A.buxifolia stems,and its proliferation coefficient was 8.8 when the concentration of TDZ was 0.50mg/L.Light culture was more conducive to the growth of thin-layer cells of A.buxifolia.The optimal adventitious bud proliferation medium was MT + 0.5 mg/L 6-BA + 2 mg/L GA3 + 0.5 mg/L IAA,the proliferation coefficient was 3.58,and the most suitable medium for bud elongation was MT + 0.1 mg/L 6-BA + 2 mg/L GA3 + 0.5 mg/L IAA,with an average plant height of 1.18 cm.The most suitable medium for adventitious root induction was 1/2 MT + 0.05 mg/L IBA + 0.4 mg/L NAA,with a rooting rate of80%.The average root number was 2.78/explant,and the average root length was 1.03 cm.The most suitable culture substrate for transplanting of cultured seedlings of A.buxifolia was a mixed substrate with a volume ratio of 6:3:1 of peaty soil,perlite and vermiculite,and the survival rate of transplanting was 81.82%.(2)Preliminary establishment of the regeneration system of A.buxifolia and preliminary research on the genetic transformation system through the somatic embryonic development pathway.The medium composed of MT + 1 mg/L GA3 + 40mg/L adenine sulfate,could induce callus of A.buxifolia,with an induction rate of5.88%.The optimal medium for embryonic callus proliferation was MT + 0.01 mg/L NAA + 2 mg/L TDZ,with the proliferation coefficient of 11.23.The optimal succession cycle for calluses in A.buxifolia was 18 d.Embryonic calluses can be induced by MT+ 3% glycerol.During the genetic transformation of A.buxifolia,Agrobacterium tumefaciens cultured for 6-10 h was the most suitable for infection.Concentrations of50 mg/L with kanamycin and 400 mg/L with cephalosporins 400 mg/L were used as a screening concentration for the genetic transformation of A.buxifolia.At present,no positive plants have been obtained,and further studies are needed. |
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