Establishment Of Callus-derived Regeneration And Study Of Agrobacterium-mediated Transformation In Casuarina Equisetifolia

Trees of Casuarina genera are widely cultivated along tropical and subtropical coastal areas as windbreak shelterbelt.Casuarina equisetifolia?C.equisetifolia?is the first one that had recently been genome-sequenced among the genera of Casuarina,which made it an ideal model tree specie for theoretic research of salinity resistance.However,the gene transformation of C.equisetifolia has not been established,which is essentially required for the molecular level research of C.equisetifolia.In this study,we used young stem shoots as material and developed a rapid and efficient callus regeneration system.Based on this regernation platform,a Agrobacterium-mediated genetic transformation method were preliminarily established.Meanwhile,we analyzed the single-base mismatch of sgRNA sequence in CRISPR/Cas9 system,which would provide guidiance not only for the design of sgRNA of Casuarina equisetifolia but also for general plant speceies.The main research results are listed as below.1.Callus regeneration system.?1?Two-steps callus indction:callus induction with 0.1 mg·L^-1 NAA,0.1 mg·L^-1 TDZ,1/2MS basal medium;callus proliferation for 10-15 days with 0.5 mg·L^-1 6-BA,0.1 mg·L^-1 TDZ,1/2MS basal medium.The callus proliferation rate was 100%.?2?Usage of 0.5 mg·L^-1 6-BA to induce gerneration of adventitious buds under light condition.?3?Root gerneration were inducted with 0.04 mg·L^-1 IAA and 0.02 mg·L^-1 IBA under light when adventitious buds were about 1cm.Root-gerneration rate was 83.25%.2.Agrobacterium tumefaciens-mediated genetic transformation.The reporter gene GUS was transformated using different strains of Agrobacteria.A series of conditions including co-culturing time,acetosyringone concentration and antibiotic selection pressures were tested.The main results are listed as follows.?1?Using C58C1 as a transformed strain.The transformation efficiency was higher after two days of co-cultivation;?2?In the hygromycin resistance screening system,20 mg·L^-1 As and 5 mg·L^-1 Hyg were used for transformation screening.GUS staining test showed that the induction rate of resistant callus was 80%.The resistance-free bud induction rate was 91.3%;?3?In the kanamycin resistance screening system,15 mg·L^-1 As and 60 mg·L^-1 Kan were initially identified for transformation screening.A higher rate of resistance callus induction was obtained,which was 76.58%.3.Analysis of sgRNA conserved region of gene knockout in CRISPR/Cas9 system:CRISPR/Cas9 with a mismatch at 20^th base?counting from the distal of PAM?of the target sites could still lead to high-frequency gene editing in rice.