红豆越橘叶片不定芽再生与VcANS基因遗传转化

发布时间：2018-04-17 01:07

  本文选题：红豆越橘 + 组织培养　； 参考：《吉林农业大学》2017年硕士论文

【摘要】：本试验以3个红豆越橘品种‘艾达’、‘桑那’和‘罗西’试管苗为材料,研究了培养基与5种植物生长物质对叶片诱导不定芽的影响,初步建立了红豆越橘离体叶片不定芽诱导体系。初步建立叶盘法将VcANS基因转至红豆越橘‘艾达’的遗传转化的实验条件,旨在为红豆越橘离体叶片不定芽诱导和遗传转化技术体系提供依据。研究结果如下:1.以不同品种的红豆越橘茎段叶片为实验材料,通过比较WPM、MS、1/2MS和1/4MS四种培养基中,最适培养基为WPM培养基;‘艾达’、‘桑那’和‘罗西’三个品种离体叶片再生体系生长调节物质最优组合为:WPM+ZT 4.0 mg/L+IBA 0.1 mg/L。‘艾达’在TDZ 2.0 mg/L时诱导率最高为99%,在ZT 5.0 mg/L的处理条件下再生频率最高为35.2个/叶;‘桑那’在TDZ 2.0 mg/L时诱导率最高为93%,在ZT 4.0 mg/L+IBA 0.1 mg/L时生频率最高为28.3个/叶;‘罗西’在ZT 4.0 mg/L+NAA 0.5 mg/L时诱导率最高为87.5%,在ZT 5.0 mg/L处理条件下再生频率最高为29.9个/叶。2.红豆越橘‘艾达’在各类生长调节物质中生长状态最佳,因此,以‘艾达’继代苗叶片为受体材料进行后续遗传转化研究。3.成功构建植物表达载体pBASTA-VcANS。4.为红豆越橘遗传转化体系筛选的抑菌剂头孢噻肟钠(Cef)最适浓度为250 mg/L;红豆越橘遗传转化阳性植株筛选试剂为除草剂草铵膦,最适使用浓度为0.9 mg/L,使用以农杆菌介导的叶盘法进行遗传转化试验,将VcANS基因转化到‘艾达’基因组中,转化体系为:将培养25~30 d的‘艾达’中部叶片,用解剖刀在叶片的近轴面上用横向划两刀处理以后平铺于预培养基中暗培养3 d后,将叶片小心取出侵入含植物表达载体pBASTA-VcANS的农杆菌菌液中,浸染10 min,浸染结束后将叶片捞出吸干多余菌液后,平铺于共培养基上,25℃暗培养2 d;用液体培养基(WPM+ZT 4.0 mg/L+IBA 0.1 mg/L+AS100 mg/L+3%蔗糖)漂洗后,将叶片转接入选择培养基(WPM+ZT 4.0 mg/L+IBA 0.1 mg/L+8g/L琼脂粉+3%蔗糖+250mg/LCef+草铵膦0.9 mg/L)中,暗培养2 d后转至光下培养,30 d后将长出的不定芽接入抗性培养基(WPM+ZT 4.0 mg/L+IBA 0.1 mg/L+8 g/L琼脂粉+3%蔗糖+250 mg/L Cef)中继代培养。5.使用农杆菌介导的叶盘法遗传转化法中,在选择条件草铵膦存在条件下,得到20棵草铵膦抗性植株,经过对它们分别进行PCR检测,结果得到一株转化植株。
[Abstract]:The effects of culture medium and five plant growth substances on adventitious buds induced by leaves were studied by using three red bean blueberry varieties' Adaganthus' sauna 'and' Rossi'in vitro.The adventitious bud induction system of red bean blueberry leaves in vitro was established.The experiment condition of transferring VcANS gene to 'Ada' by leaf disk method was established to provide the basis for adventitious bud induction and genetic transformation of red bean blueberry leaves in vitro.The results are as follows: 1.The stem leaves of different varieties of red bean blueberry were used as experimental materials. By comparing the four kinds of media, WPMN MS 1 / 2 MS and 1/4MS, the most suitable medium was WPM medium.The optimum combination of growth regulators of 'sauna' and 'Rossi' leaves in vitro was: 1: WPM ZT 4.0 mg/L IBA 0.1 mg / L.Under the condition of ZT 5.0 mg/L, the highest induction rate of 'Ida' was 93.30 at TDZ 2.0 mg/L, and that of ZT 4.0 mg/L IBA 0.1 mg/L was 28.3 / leaf.The highest induction rate of 'Rossi' was 87.5 at ZT 4.0 mg/L NAA 0.5 mg/L, and the highest regeneration frequency was 29.9 leaves / leaf under the condition of ZT 5.0 mg/L.Red bean blueberry 'Ada' is the best growth regulator among all kinds of growth regulators. Therefore, the following genetic transformation study was carried out with the leaves of 'Ada' subculture seedling as the recipient material. 3.The plant expression vector pBASTA-VcANS.4was successfully constructed.The optimum concentration of cefotaxime sodium cefin for screening genetic transformation system of red bean blueberry was 250 mg / L.The optimal concentration was 0.9 mg / L, and Agrobacterium tumefaciens-mediated leaf disc method was used to carry out genetic transformation test. The VcANS gene was transformed into the genome of 'Ada'. The transformation system was as follows: the middle leaf of 'Ida' was cultured for 2530 days.The leaves were treated with two transversal blades on the paraxial surface of the leaves and cultured in a dark culture medium for 3 days. The leaves were carefully removed and invaded into Agrobacterium tumefaciens solution containing plant expression vector pBASTA-VcANS.After soaking for 10 minutes, the leaves were removed from the superfluous bacteria solution after soaking, and then spread on the co-culture medium at 25 鈩，

本文编号：1761372