叠氮化钠及甲氧咪草烟对水稻幼胚愈伤组织成活、丛生芽分化及生根的影响

Effects of sodium azide and methotrexate on callus survival,cluster bud differentiation and rooting of immature embryos of rice

目的:研究不同浓度叠氮化钠(NaN_(3))及甲氧咪草烟(Methotrexate)对水稻幼胚愈伤组织成活、丛生芽分化及生根的影响。方法:以优质水稻品种‘润珠香占’幼胚愈伤组织为外植体,用1、2、3、4 mmol/L叠氮化钠处理水稻幼胚愈伤组织8 min后,将其分别接种到含0.001、0.005、0.010、0.050 g/L甲氧咪草烟的愈伤组织生长培养基中,观察愈伤组织生长状况,统计愈伤组织褐化率、成活率,再移入分化生根培养基中,统计成活愈伤组织分化率、生根率。结果:水稻愈伤组织甲氧咪草烟致死质量浓度为0.05 g/L。甲氧咪草烟是影响水稻幼胚愈伤组织成活的重要因子,其次是叠氮化钠,再次为它们的交互作用,处理间差异显著。随着叠氮化钠、甲氧咪草烟质量浓度的增加,水稻愈伤组织由白色变为淡黄色,直至褐色,褐化率逐渐增加,愈伤组织成活率逐渐减小,A_(1)、A_(2)、A_(3)与B_(1)、B_(2)、B_(3)各处理组合,水稻幼胚愈伤组织呈白色或淡黄色,褐化率较小,为6.67%~50.00%,成活率较大,为23.33%~96.67%;A_(4)与B_(1)、B_(2)、B_(3)、B_(4)各处理组合,愈伤组织大部分或全部褐化,褐化率较大,为86.67%~100.00%,成活率较小,为0.00%~6.67%;B_(4)与A_(1)、A_(2)、A_(3)、A_(4)各处理组合,愈伤组织褐化较多,褐化率较大,为66.67%~100.00%,成活率较小,为0.00%~16.67%。随着NaN_(3)、甲氧咪草烟质量浓度的增加,水稻愈伤组织丛生芽分化率有逐渐减少的趋势,处理组合间差异显著,其中A_(1)B_(2)、A_(2)B_(2)分化率最高,A_(4)B_(2)有部分分化,但较低,分化率只有5.56%;A_(4)B_(1)、A_(4)B_(3)不能分化出丛生芽。丛生芽经培养,生根率均达100.00%,无统计学意义。结论:NaN_(3)处理的愈伤组织在0.05 g/L甲氧咪草烟中培养,有0.00%~6.67%的成活率,与对照相比降低90.00%~96.67%,并有5.56%成活愈伤组织能分化出丛生芽并生根,即A_(4)B_(2)组合(0.05 g/L甲氧咪草烟、2 mmol/L NaN_(3))为诱导水稻幼胚愈伤组织抗甲氧咪草烟变异、成活、丛生芽分化及生根的最佳处理组合,可为今后水稻抗除草剂育种奠定一定的理论和实践基础。

Objective:To study the effects of different concentrations of NaN_(3)and methotrexate on the survival,cluster bud differentiation and rooting of immature embryo callus in rice(Oryza sativa L.).Methods:The immature embryo callus of high-quality rice variety Runzhuxiangzhan was used as explants.The calli of rice immature embryos were treated with 1,2,3,4 mmol/L NaN_(3)for 8 min,and they were inoculated into the callus growth medium containing 0.001,0.005,0.010,0.050 g/L of methotrexate respectively.The growth status of callus was observed,the browning rate and survival rate of callus were calculated,and then the differentiation rate and rooting rate of callus were calculated.Results:The lethal concentration of methotrexate in rice callus was 0.05 g/L.Methotrexate was an important factor affecting the survival of immature embryo callus in rice,followed by NaN_(3),and then for their interaction.The difference between treatments was significant.With the increase of the concentration of NaN_(3)and methotrexate,the callus of rice changed from white to light yellow and brown,the browning rate increased gradually,and the survival rate of callus decreased gradually.A_(1),A_(2),A_(3)and B_(1),B_(2),B_(3)treatment combinations,young embryos of rice callus were white or light yellow,brown rate was 6.67%—50.00%,the survival rate was 23.33%—96.67%.The browning rate was 86.67%—100.00%and the survival rate was 0.00%—6.67%in the combination of A_(4)and B_(1),B_(2),B_(3),B_(4).The combination of B_(4)and A_(1),A_(2),A_(3),A_(4),the callus browning was more,the browning rate was 66.67%—100.00%,and the survival rate was 0.00%—16.67%.With the increase of the concentration of NaN_(3)and methotrexate,the rate of cluster bud differentiation of rice callus decreased gradually,and there was significant difference among the treatments.Among them,A_(1)B_(2)and A_(2)B_(2)had the highest rate of differentiation,A_(4)B_(2)had partial differentiation,but the lower,the rate of differentiation was only 5.56%,A_(4)B_(1)and A_(4)B_(3)could not differentiate into buds.The rooting rate of clustered buds was 100.00%after culture,with no statistical difference.Conclusion:The survival rate of NaN_(3)-treated callus cultured in 0.05 g/L methotrexate was 0.00%—6.67%,compared with the control decreased by 90.00%—96.67%and 5.56%of the surviving callus could differentiate into cluster buds and take root.The combination of A_(4)B_(2)(0.05 g/L methotrexate and 2 mmol/L NaN_(3))was the combination to induce the mutation survival,bud differentiation and rooting of rice immature embryos callus.It laid a certain theoretical and practical foundation for rice herbicide resistance breeding in the future.