根癌农杆菌介导彩色马蹄莲遗传转化体系的建立及优化  

Establishment and optimization of Agrobacterium tumefaciens mediated genetic transformation system for Zantedeschia hybrida Spr.

作　　者：李菲 曹永琼[2] 王纲 郭彦兵[1] 李紫薇 吴红芝[1] LI Fei;CAO Yong-qiong;WANG Gang;GUO Yan-bing;LI Zi-wei;WU Hong-zhi(College of Landscape and Horticulture,Yunnan Agricultural University,Kunming,Yunnan 650201,China;Office ofScience and Technology,Kunming University,Kunming,Yunnan 650214,China)

机构地区：[1]云南农业大学园林园艺学院,云南昆明650201 [2]昆明学院科学技术处,云南昆明650214

出　　处：《南方农业学报》2024年第6期1692-1699,共8页Journal of Southern Agriculture

基　　金：国家自然科学基金项目(32160723,31960610)。

摘　　要：【目的】建立及优化根癌农杆菌介导的彩色马蹄莲(Zantedeschia hybrida)遗传转化体系,为提高彩色马蹄莲遗传转化效率及培育彩色马蹄莲抗性品种提供理论参考依据。【方法】将不同外植体(茎基、叶、不定芽)和不同浓度噻苯隆(TDZ)(0.001、0.002和0.005 g/L)进行正交试验筛选外植体和丛生芽增殖培养基;液氮冻融法将pCAM BIA2301和pBI121质粒转入农杆菌LBA4404感受态细胞。以β-葡萄糖苷酸酶(GUS)基因作为农杆菌介导转化测定的报告基因,对彩色马蹄莲的丛生芽进行遗传转化,阳性苗经PCR扩增验证转化结果。通过不同硫酸卡那霉素(Kan)浓度(50、100和150 mg/L)、侵染时间(25、30和40 min)及共培养时间(2和3 d)3因素正交试验筛选最佳转化条件。【结果】外植体为不定芽的丛生芽诱导率极显著高于茎基和叶(P<0.01),当不定芽为外植体、TDZ浓度为0.002 mg/L时,丛生芽增殖倍数(5.12倍)显著高于0.001和0.005 mg/L TDZ处理(P<0.05),筛选出M6培养基+0.002 mg/L TDZ+30 g/L蔗糖+6.25 g/L琼脂为丛生芽增殖培养基;影响阳性频率因素排序为:侵染时间>Kan浓度>共培养时间。当Kan浓度为50 mg/L、侵染时间25 min及共培养时间为3 d为最优侵染条件,此时,GUS+丛生芽出芽频率和PCR阳性频率最高,分别为54.0%和15.27%。【结论】成功优化彩色马蹄莲遗传转化体系,最佳外植体为不定芽,转化体系最佳组合为Kan浓度为50 mg/L、侵染时间为25 min及共培养时间为3 d。【Objective】This study aimed to establish and optimize an Agrobacterium tumefaciens mediated genetic transformation system for Zantedeschia hybrida Spr.,providing theoretical support for improving the efficiency of genetic transformation and cultivating resistant varieties of Z.hybrida Spr.【Method】An orthogonal experiment was conducted using different explants(stem base,leaves,and adventitious buds)and different concentrations thidiazuron(TDZ)of 0.001,0.002 and 0.005 mg/L to determine the optimal explant and clustered bud proliferation medium.The pCAM BIA2301 and pBI121 plasmids were introduced into A.tumefaciens LBA4404 competent cells using the liquid nitrogen freeze-thaw method.The Z.hybrida Spr.clustered buds were genetically transformed usingβ-glucuronidase(GUS)gene as a reporter gene for A.tumefaciens mediated transformation.Positive seedlings were verified by PCR amplification.The optimal transformation conditions were screened by orthogonal experiment with three factors:kanamycin(Kan)concen trations(50,100 and 150 mg/L),infection time(25,30 and 40 min),and co-culture time(2 and 3 d).【Result】The in duction rate of clustered buds using adventitious bud as an explant was extremely significantly higher than that from stem base and leaf(P<0.01).When adventitious bud was used as explant and the TDZ concentration was 0.002 mg/L,the pro liferation times of clustered buds(5.12 times)was significantly higher than that of 0.001 and 0.005 mg/L TDZ treatments(P<0.05).The optimal medium for clustered bud proliferation was M6 medium+0.002 mg/L TDZ+30 g/L sucrose+6.25 g/L agar.The factors affecting the positive transformation frequency were ranked as:infection time>Kan concentration>co�culture time.The ideal infection conditions were:50 mg/L Kan,25 min infection time,and 3 d co-culture time,resulting in the highest GUS+clustered bud emergence frequency(54.0%)and PCR positive frequency(15.27%).【Conclusion】An efficient A.tumefaciens mediated genetic transformation system for Z.hybrida Spr.is successfully

关 键 词：彩色马蹄莲 根癌农杆菌 遗传转化 优化 

分 类 号：S682.264[农业科学—观赏园艺]