外源褪黑素对木薯脆性胚性愈伤组织增殖的影响

Effect of exogenous melatonin on the proliferation of friable embryogenic callus in cassava

以木薯(Manihot esculenta)‘TMS60444’脆性胚性愈伤组织(FEC)为材料,研究了在GD基础培养基中添加不同浓度外源褪黑素(melatonin)对FEC增殖的影响。结果表明:在不含氨氯吡啶酸(4-氨基-3,5,6-三氯吡啶-2-羧酸)的GD培养基(GD0)中加入不同浓度褪黑素,其中低浓度(<100μmol·L^(-1))褪黑素可以促进FEC增殖,但高浓度褪黑素使FEC生长缓慢并褐化。在含有12 mg·L^(-1)氨氯吡啶酸的GD培养基(GD1)中添加不同浓度褪黑素(0、0.1、1、10、100μmol·L^(-1))进行培养,FEC鲜重增长率明显不同;以添加10μmol·L^(-1)褪黑素效果最佳,可使FEC保持较好的增殖状态,并显著提高木薯FEC鲜重的增长率,比不加褪黑素的GD1-1培养基中增加了2.57倍。用10μmol·L^(-1)褪黑素处理木薯FEC,其内源褪黑素代谢通路相关基因表达发生改变,抗氧化相关基因明显上调表达;并且抗氧化酶活性显著增强,抗氧化能力明显提高。本研究为木薯遗传转化中延长木薯FEC使用周期和提高增殖效率提供思路和技术。

To investigate the effect of melatonin on friable embryogenic callus(FEC)proliferation in cassava(Manihot esculenta)‘TMS60444',GD media with different melatonin levels were tested for FEC subculturing.When GD media was without picloram(4-amino-3,5,6-trichloropyridine-2-carboxylic acid)(GD0),supplementing with low concentrations of melatonin(less than 100μmol·L^(-1))showed a positive effect on FEC proliferation.Meanwhile,FEC showed inhibited growth and browning in the GD0 medium with higher levels of melatonin.When GD media was supplemented with 12 mg·L^(-1)picloram(GD1)and different levels of melatonin(0,0.1,1,10,and 100μmol·L^(-1)),the proliferation rates of FEC were variable and 10μmol·L^(-1)melatonin was the most suitable concentration for better growth,showing a 2.57-fold increase of fresh mass that of GD1-1 control.Under the 10μmol·L^(-1)melatonin treatment,there was altered expression in melatonin biosynthetic genes and upregulated expression of antioxidant enzyme genes in FEC,leading to improved antioxidant capacity by increased ROS-scavenging enzymatic activity.Our study provides a practical approach for prolonging the viability of cassava FEC during genetic transformation.