烟草毛状根多倍体诱导及其植株再生

Induction of polyploid in hairy roots of Nicotiana tabacum and its plant regeneration

摘要:

为了提高烟草的烟碱含量，采用发根农杆菌遗传转化和人工染色体加倍技术，进行了烟草毛状根及其多倍体诱导、植株再生及其烟碱含量测定。结果表明，发根农杆菌ATCC15834感染烟草叶片外植体8 d后产生白色毛状根，15 d后所有叶片外植体产生毛状根。毛状根能在无外源激素的 MS固体和液体培养基上自主生长。PCR扩增结果显示发根农杆菌Ri质粒的rol B和rol C 基因以及冠瘿碱合成酶基因已在烟草毛状根基因组中整合并得到表达。烟草毛状根多倍体诱导的最适条件为0.1%的秋水仙素溶液处理36 h，其多倍体诱导率为64.71%。经秋水仙素加倍的烟草毛状根多倍体植株再生的最适宜培养基为MS+6-BA 2.0 mg/L+NAA 0.2 mg/L。与对照 (二倍体非转化植株) 相比，烟草二倍体毛状根再生植株的顶端优势减弱，腋芽增多，叶片变窄；而烟草毛状根多倍体再生植株茎更粗，节间变短，叶色更深，叶片的宽度和厚度均较对照明显增大。根尖细胞染色体压片观察证实，所获得的烟草毛状根多倍体再生植株为四倍体，其根尖细胞染色体数约为4n=96。盆栽实验表明, 烟草二倍体毛状根植株和多倍体毛状根再生植株比对照植株延迟约 21 d开花。GC-MS检测表明，烟草毛状根多倍体再生植株的烟碱含量比对照及二倍体毛状根再生植株显著提高，分别约为对照及二倍体毛状根再生植株的6.90倍和4.57倍。

Abstract:

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.