| 红比利时杜鹃愈伤悬浮颗粒瞬时转化体系构建及植株再生 |
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| 引用本文： | 何凡,吴月燕,谢晓鸿,杨国霞,蒋宝鑫,李东宾,鄢毅铖,贾永红.红比利时杜鹃愈伤悬浮颗粒瞬时转化体系构建及植株再生[J].核农学报,2022,36(8):1546-1558. |
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| 作者姓名： | 何凡 吴月燕 谢晓鸿 杨国霞 蒋宝鑫 李东宾 鄢毅铖 贾永红 |
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| 作者单位： | 1浙江万里学院生物与环境学院,浙江 宁波 3151002宁波市林场,浙江 宁波 315440 |
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| 基金项目： | 浙江省重点研发计划(2021C02053);宁波市2021年科技创新2025现代种业重大专项(2021Z005);宁波市科技特派员项目(202003N6002);省一流学科学生创新项目(SZ1000011005045) |
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| 摘 要： | 为建立红比利时杜鹃花愈伤悬浮颗粒瞬时转化体系,以嫩叶为材料,探讨了愈伤组织诱导与增殖的条件,构建并评价了愈伤悬浮颗粒瞬时转化体系,同时诱导愈伤颗粒出芽、生根,并获得组培小苗。结果表明,在2.41 g·L-1木本植物基本培养基(WPM)+20 g·L-1蔗糖+10 g·L-1麦芽糖+7.0 g·L-1琼脂+0.050 mg·L-1植物组培抗菌剂(PPM)为基本培养基的条件下,嫩叶愈伤组织诱导的生长调节剂最优方案为:0.3 mg·L-1 2,4-二氯苯氧乙酸(2,4-D)+0.3 mg·L-1 噻重氮苯基脲(TDZ),此时愈化率可达97.78%;愈伤组织继代增殖的生长调节剂方案为:0.61 mg·L-12,4-二氧苯氧乙酸(2,4-D)+0.65 mg·L-1 6-苄基腺嘌呤(6-BA)+1.37 mg·L-1 TDZ,此时增殖率可达167%。将继代5次后呈疏松状的愈伤颗粒接种于液体继代培养基中,大约4~12 d后愈伤颗粒进入指数生长阶段,增殖率为45%。含有GUS基因的pRI 101-AN与含有GFP基因的pCAMBIA1301-GFP分别在农杆菌GV3101介导下转化处于指数生长期的愈伤悬浮颗粒,OD600为0.6的农杆菌侵染液侵染15 min后,愈伤颗粒的GUS染色效率为26.28%,愈伤颗粒的GFP荧光表达效果明显。另外,以2.41 g·L-1 WPM+7 g·L-1琼脂+20 g·L-1 蔗糖为基本培养基,添加2.0 mg·L-1TDZ + 0.5 mg·L-1 2,4-D时,可诱导愈伤组织不定芽;以1/2 Read为基本培养基,添加0.5 mg·L-1 IBA+1 g·L-1活性炭可诱导出芽的愈伤颗粒生根。本研究结果为进一步开展红比利时杜鹃花稳定遗传转化研究和转基因植株培育奠定了基础。
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| 关 键 词： | 红比利时杜鹃 愈伤悬浮颗粒 瞬时转化 再生 |
| 收稿时间： | 2022-04-11 |
Construction of Transient Transformation System of Callus Suspension Particle From Rhododendron × hybridum Hort and Plant Regeneration |
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| HE Fan,WU Yueyan,XIE Xiaohong,YANG Guoxia,JIANG Baoxin,LI Dongbin,YAN Yicheng,JIA Yonghong.Construction of Transient Transformation System of Callus Suspension Particle From Rhododendron × hybridum Hort and Plant Regeneration[J].Acta Agriculturae Nucleatae Sinica,2022,36(8):1546-1558. |
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| Authors: | HE Fan WU Yueyan XIE Xiaohong YANG Guoxia JIANG Baoxin LI Dongbin YAN Yicheng JIA Yonghong |
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| Institution: | 1College of Biological & Environmental Sciences, Zhejiang Wanli University, Ningbo, Zhejiang 3151002Ningbo Forest Farm, Ningbo, Zhejiang 315440 |
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| Abstract: | In order to establish the transient transformation system of Rhododendron × Hybridum Hort, the young leaves were used as materials to explore the conditions of callus induction and proliferation, the transient transformation system of the callus suspension particles was constructed and evaluated, along with the induction of budding and roots until obtaining the seedling. The results showed that, under the conditions of 2.41 g·L-1 WPM + 20 g·L-1 sucrose + 10 g·L-1 maltose + 7.0 g·L-1 agar + 0.050 mg·L-1 PPM antimicrobial as the basic medium, the growth regulators for callus induction of young leaves were 0.3 mg·L-1 2, 4-D + 0.3 mg·L-1 TDZ, the healing rate can reach about 97.78%. For the sub-proliferation of callus the growth regulator was 0.61 mg·L-1 2, 4-D + 0.65 mg·L-1 6-BA + 1.37 mg·L-1 TDZ, and the proliferation rate could reach 167%. After 5 subcultures, the loose callus particles were inoculated into the liquid subculture medium. 4~12 days later, the callus particles entered the exponential growth stage, and the proliferation rate was about 45%. Under the mediation of Agrobacterium tumefaciens strain GV3101, the pRI 101-AN containing GUS gene and the pCAMBIA1301-GFP containing GFP gene were respectively transformed into callus in exponential growth phase, the GUS staining efficiency of callus particles was 26.28% and the GFP fluorescence expression effect of callus particles was remarkable when the OD600 infection solutions was 0.6 and infected time was 15 min. In addition, under the conditions of 2.41 g·L-1 WPM + 7.0 g·L-1 agar + 20 g·L-1 sucrose as the basic medium, the adventitious bud was induced from callus particles with the addition of growth regulator of 2.0 mg·L-1 TDZ + 0.5 mg·L-1 2, 4-D, of which was transferred to 1/2 Read basic medium with the addition of growth regulator of 0.5 mg·L-1 IBA + 1 g·L-1 activated carbon to induce the adventitious root. The study laid a foundation for further research on the stable genetic transformation and the cultivation of transgenic plant of Rhododendron × hybridum Hort. |
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| Keywords: | Rhododendron × hybridum Hort callus suspension particles transient transformation regeneration |
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