| Chinese chives (Allium tuberosum Rottler)which is riched in nutritious and has many health functions. But traditional breeding of chinese chive is slow and natural populations is lack of effective resistance gene. With the increasing maturity of transgenic technology, the application of modern biotechnology breeding of new varieties has been a great success. Therefore, using "Hanzhong Dongjiu" variety as the material, the paper established a stable and efficient regeneration system from the radical and observed the regeneration of adventitious buds during various periods in histology; In addition, based on the regeneration system, the factors related to Agrobacterium-mediated genetic transformation were researched by the Agrobacterium EHA105containing pCAMBIA3301plasmid infecting the callus of chinese chive. The results were presented as below.1. The different parts of the radicle of chinese chive were used as explants to induce callus and the single plants were differentiated in different hormones and concentration combination media including NAA, IAA,6-BA, KT and GA. The results showed that the radicle tip is the best explants for callus induction and the callus induction rate is81.4%, the bud induction rate reach73.3%. The regeneration rate was the highest in MS+1mg/L BA+0.5mg/L NAA+0.5mg/L KT medium, the callus induction and bud differentiation rate were up to more than80%and78.8%respectively, the propagation coefficient was24. The percentage of adventitious buds elongating and become single plant was85.5%in MS+1mg/L GA+0.5mg/L KT and the plant reached7cm in height. For single plant rooting, the best medium was1/2MS+0.3mg/L IAA and the root grew very well.2. Using the callus and adventitious buds obtained by tissue culture as the material, the paper researched the staining method for paraffin section of chinese chive and observed the differentiation stage of chinese chive with the best staining method. The experimental results showed that staining the tissues for8h with safranine and20s with Fast Green is very good and the tissue is stained fully. The color of the tissue showed bright after stained for4h and8h with safranine and showed dark green after stained for20s with fast green. When the callus and bud of chinese chive developed, firstly the radicle tip de-differentiated and formed callus, then the callus began to re-differentiated and adventitious buds began to re-differentiated from the callus and grew till to become a single plant with bulb.3. Using the callus of chinese chive as the explant for Agrobacterium-mediated transformation, the paper studied the factors which influence transformation efficiency of chinese chive, and meantime screened the concentration of PPT and Cef. The results showed that the concentration of PPT and Cef were1mg/L and400mg/L at the callus induction and adventitious bud differentiation stage, while the concentration were0.5mg/L and200mg/L at the rooting stage. When the callus cultured for40days was used for genetic transformation, GUS transient expression rate reached93%and the adventitious buds differentiated well; When100μmol/L acetosyringone was added to the miedium, GUS expression rate of the callus reached91%and the plant regeneration rate was7.9%. They did not increase with increasing of the AS concentration; Compared to other combinations, the calli were soaked in Agrobacterium(OD600≈0.6) for10min, the injury degree of explants was slight and the GUS expression rate and regeneration rate were the highest; When the calli were co-cultured for3days after infection, the bacterium grew less than other period, GUS expression rate and the regeneration rate were91.1%and7.2%respectively. This provided some references for genetic transformation of chinese chive in the future. |
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