Abstract: Chicory(Cichorium intybus L.cv.Puna) leaf segments from aseptic seedlings were used as experimental materials.The explants were inoculated onto the MS medium with various phytohormone combinations to induce callus formation,and bud and root regeneration.Effects of phytohormone concentrations and combinations on the induction of callus,buds and roots were analyzed.Agrobacterium tumefaciens LBA4404(harboring plasmid pBin438-TaNHX2) was used to infect Puna Chicory explants based on the regeneration system that had been established for the high efficiency transformation of the cultivar.Result showed that both suitable pre-culture time and co-culture time of explants with Agrobacterium tumefaciens were 2~3 days.The infection time was 8 minutes and Kanamycin concentration used for selecting positive regenerated buds was 60 mg·L^-1.Effects of acetosyringone(AS) and ultrasonication on the rate of Agrobacterium-mediated transformation were also investigated.Results showed that acetosyringone of 200 μmol·L^-1 was the optimum concentration for enhancing Agrobacterium transformation efficiency,and ultrasonication treatment of 200 W and negative pressure of 20 times also improved the transformation rate.The maximum kanamycin concentration for wild type Puna chicory survival was 26 mg·L^-1;and the suitable concentration of Cefazolin sodium,cefotaxime sodium,carbenicillin and Benzylpenicillin for inducing callus formation and bud differentiation were 500~1000,500~1000,300 and 40~60 mg·L^-1,respectively.The vacuolar Na⁺/H⁺ exchanger gene(TaNHX2) was introduced into the chicory cultivar,and 28 transgenic plants were obtained by PCR detection and Southern blot analysis.

Key words: Puna chicory, Tissue culture, Plant regeneration, Agrobacterium-mediated, Genetic transformation, TaNHX2

摘要： 以普那菊苣(Cichorium intybus L.cv.Puna)叶片为试验材料,接种于含不同激素浓度配比的MS培养基上进行愈伤组织、芽分化以及根再生的诱导,分析了不同激素浓度及其配比对愈伤组织诱导和芽分化以及根再生效果的影响。以已经建立的再生体系为基础,以农杆菌菌株LBA4404(含质粒pBin438-TaNHX2)侵染转化普那菊苣,探索普那菊苣高效遗传转化体系。结果表明:对外植体适宜的预培养时间为2～3d,与农杆菌的共培养时间也应控制在2～3d;侵染时间控制在8min左右;卡那霉素(Km)阳性筛选的适宜选择浓度为60mg·L^-1。乙酰丁香酮(AS)200μmol·L^-1是促进农杆菌转化的最佳浓度,200 W超声波处理、20次负压处理也可提高农杆菌转化率效果。26mg·L^-1 Km是野生型普那菊苣苗能够存活的上限,头孢唑林钠和头孢噻肟钠在500～1000mg·L^-1浓度范围内、羧苄青霉素300mg·L^-1和氨苄青霉素在40～60mg·L^-1浓度范围内均能较好的诱导出愈伤组织和芽。将来自小麦(Triticum aestivum)的Na⁺/H⁺逆向转运蛋白(vacuolar Na⁺/H⁺exchanger or antiporter,简称NHX,NHE或NHA)导入普那菊苣;经抗生素筛选以及针对TaNHX2基因的PCR检测和Southern杂交分析,证明获得了28株转TaNHX2基因的普那菊苣植株。

关键词: 普那菊苣, 组织培养, 植株再生, 农杆菌介导, 遗传转化, TaNHX2