煙草毛狀根多倍體誘導及其植株再生

為了提高煙草的菸鹼含量，採用髮根農桿菌遺傳轉化和人工染色體加倍技術，進行了煙草毛狀根及其多倍體誘導、植物再生及其菸鹼含量測定。結果表明，髮根農桿菌ATCC15834感染煙草葉片外植體8 d後產生白色毛狀根，15 d後所有葉片外植體產生毛狀根。毛狀根能在無外源激素的MS固體和液體培養基上自主生長。PCR擴增結果顯示髮根農桿菌Ri質粒的rol B和rol C基因以及冠癭鹼合成酶基因已在煙草毛狀根基因組中整合併得到表達。煙草毛狀根多倍體誘導的最適條件為0.1%的秋水仙素溶液處理36 h，其多倍體誘導率為64.71%。經秋水仙素加倍的煙草毛狀根多倍體植株再生的最適宜培養基為MS+6-BA 2.0 mg/L+NAA 0.2 mg/L。與對照(二倍體非轉化植株)相比，煙草二倍體毛狀根再生植株的頂端優勢減弱，腋芽增多，葉片變窄；而煙草毛狀根多倍體再生植株莖更粗，節間變短，葉色更深，葉片的寬度和厚度均較對照明顯增大。根尖細胞染色體壓片觀察證實，所獲得的煙草毛狀根多倍體再生植株為四倍體，其根尖細胞染色體數約為4n=96。盆栽實驗表明，煙草二倍體毛狀根植株和多倍體毛狀根再生植株比對照植株延遲約21 d開花。GC-MS檢測表明，煙草毛狀根多倍體再生植株的菸鹼含量比對照及二倍體毛狀根再生植株顯著提高，分別約為對照及二倍體毛狀根再生植株的6.90倍和4.57倍。
By genetic transformation with Agrobacterum rhizogenes and artificial chromosome doubling techniques, we studied the induction of hairy roots and their polyploidization, and subsequent plant regeneration and nicotine determination to enhance the content of nicotine in Nicotiana tabacum. The results show that hairy roots could be induced from the basal surface of leaf explants of N. tabacum 8 days after inoculation with Agrobacterium rhizogenes ATCC15834. The percentage of the rooting leaf explants was 100% 15 days after inoculation. The hairy roots could grow rapidly and autonomously on solid or liquid phytohormones-free MS medium.The transformation was confirmed by PCR amplification of rol gene of Ri plasmid and paper electrophoresis of opines from N. tabacum hairy roots. The highest rate of polyploidy induction, more than 64.71%, was obtained after treatment of hairy roots with 0.1% colchicine for 36 h. The optimum medium for plant regeneration from polyploid hairy roots was MS+2.0 mg/L 6-BA +0.2 mg/L NAA. Compared with the control diploid plants, the hairy roots-regenerated plants had weak apical dominance, more axillary buds and more narrow leaves; whereas the polyploid hairy root-regenerated plants had thicker stems, shorter internodes and the colour, width and thickness of leaves were significantly higher than that of the control. Observation of the number of chromosomes in their root tip cells reveals that the obtained polyploid regenerated plants were tetraploidy, with 96(4n=96) chromosomes. Pot-grown experiments showed compared to the control, the flowering was delayed by 21 days in diploid hairy roots-regenerated plants and polyploid hairy root-regenerated plants. GC-MS detection shows that the content of nicotine in polyploid plants was about 6.90 and 4.57 times the control and the diploid hairy roots-regenerated plants, respectively. Copyright © 2014 中國科學院微生物研究.