景宁木兰组织培养外植体选择与抗褐化研究

Explant selecting and anti-browning of Magnolia sinostellata in tissue culture

该研究以景宁木兰(Magnolia sinostellata)为材料,从外植体类型的选择、消毒时间、预处理方法、离体培养条件和抗褐化剂类型选择等方面进行综合研究,以期筛选出景宁木兰组织培养的最佳方案。结果表明:利用0.1%氯化汞(HgCl_2)浸泡处理14和8 min分别是景宁木兰叶片和带芽茎段、根部的最佳消毒时间;景宁木兰叶片及茎段的褐化率在暗培养7 d时,分别为60.00%和56.67%,14 d时显著升高,而根部则在暗培养14 d时褐化率最低(45%);利用1 g·L^(-1)聚乙烯吡咯烷酮(PVP)浸泡预处理景宁木兰外植体6 h可显著降低(P<0.05)其褐化程度,处理后叶片、带芽茎段和根部的褐化率分别降至45.00%、28.33%和63.33%。不同的抗褐化剂均可减轻外植体的褐化程度,但针对不同外植体其效果不同。景宁木兰叶片最佳抗褐化剂为0.02 g·L^(-1)硝酸银(AgNO_3),可使其褐化率降低至16.67%,成活率为11.67%;带芽茎段和根部的最优抗褐化剂为2 g·L^(-1)柠檬酸(CA),褐化率分别降至30.00%和5.00%,成活率依次为50.00%和76.67%。综上所述,带芽茎段和根部抗褐化处理后褐化率较低且成活率较高,为景宁木兰组织培养最佳外植体。

Magnolia sinostellata is one of the endangered plants in Magnoliaceae,and is also facing the challenge of tissue culture for browning. Therefore,the leaf,stem with bud and root of M. sinostellata as explants were treated in different disinfection time（ 8,10,12,14 min）,light and dark condition,pretreated by PVP and Vc,and the basic media was MS（ Murashige and Skoog） ＋0.5 mg·L^-16-BA＋0.3 mg·L^-1IAA,pH 5.8） in this study. And then,three explantswere treated by the basic media with 0.5,1,2 g·L^-1 anti-browning substance（ Vc,PVP,CA,AgNO3,Na2S2O3） for getting the optimal tissue culture methods. All culture processes were put in 25 ℃ condition. The results showed that 14 min was the best sterilization time for the leaves and stems with bud,and 8 min was the optimal sterilization time for the roots under 0.1% HgCl2 disinfection. The browning rates of leaves and stems were the lowest at 7 d during the early stage under dark culture condition,which was 60.00% and 56.67% respectively,and the browning rate of root was 45.00% at 14 d. Soaking explants in 1 g·L^-1 PVP for 6 h as pretreatment can significantly reduce the browning rate（ P0.05）,the browning rate of leaves,stems with buds and roots were reduced to 45%,28.33% and 63.33% respectively. Anti-browning agents and concentrations can effectively reduce the degree of browning of the explants in M. sinostellata. The best anti-browning substance for leaves was 0.02 g·L^-1 AgNO3 and its browning rate reduced to 16.67%,the survival rate was 11.67%; 2 g·L^-1 CA was the optimal anti-browning substance for stems with buds and root,the browning rate were 30% and 50%,the survival rate was 50.00% and 76.67%. In conclusion,we found that the stem segment with bud and root were the best explants for tissue culture of M. sinostellata. This research had obtained the best scheme for preventing the browning of explants in M. sinostellata,which is helpful for the rapid propagation technique of M. sinostellata,and to provide reference for the study of other species of Magnoliaceae.