Improving Infection Efficiency of Agrobacterium to Immature Cotyledon Explants of Japanese Apricot (Prunus mume) by Sonication Treatment

Abstract

The infection efficiency of Agrobacterium to cotyledon explants of Japanese apricot (Prunus mume) was markedly improved by sonication treatment. The use of sGFP(S65T) reporter gene in this study enabled direct observation of transgene expression, thus allowing the evaluation of Agrobacterium infection efficiency. Immature cotyledons of different cultivars and different developmental stages were subjected to sonication treatment of durations ranging from 10 sec to 2 min. When early-stage immature cotyledons of ‘Nanko’ were subjected to sonication treatment, the transient GFP expression frequency after co-cultivation was nearly 100% and GFP fluorescence was distributed over almost the entire cotyledon surface. In contrast, when the inoculation was carried out according to the standard dipping method, transient GFP expression frequency was less than 10% and GFP fluorescence was observed only in spots. The highest frequency of somatic embryogenesis (SEG) with GFP expression was obtained from 40 sec sonication treatment of May 14 ‘Nanko’ immature cotyledons. However, with younger cotyledons, which have a higher SEG frequency, 20 sec of sonication treatment was found to be sufficient to increase the infection frequency.

ウメ未熟子葉を用いた形質転換において, アグロバクテリウムを感染する際に子葉外植体を超音波処理することにより, アグロバクテリウムの感染率が著しく増加した. 本研究では, sGFP (S65T) レポーター遺伝子を用いたため, 導入遺伝子の発現を直接観察でき, 導入遺伝子発現の評価が容易になった. 数品種の異なる発育段階のウメ未熟子葉を用いて, アグロバクテリウム菌液に浸漬した状態で 10 秒から 2 分間超音波処理を行い, その後, 感染率および GFP 発現を示す不定胚再分化率を調査した. 発育段階初期のウメ‘南高’の未熟子葉への超音波処理区では, 共存培養後 GFP の一過性発現率は 100％に近く, GFP 蛋白質の蛍光が子葉外植体の全面でみられた. しかし, 子葉外植体をアグロバクテリウム菌液に浸漬したのみの対照区では, GFP の一過性発現率は 10％未満と低く, GFP 蛍光が点もしくは限られた部位にしかみられなかった. 5 月 14 日の‘南高’ウメの未熟子葉を 40 秒間超音波処理した区で, GFP を発現する不定胚再生効率が最も高かった. しかし, 不定胚再生能がより高い, より若い未熟子葉を用いた場合, 20 秒間の超音波処理でも十分な感染率を獲得することができた.