https://www.ahs.ac.cn/images/0513-353X/images/top-banner1.jpg|#|苹果
https://www.ahs.ac.cn/images/0513-353X/images/top-banner2.jpg|#|甘蓝
https://www.ahs.ac.cn/images/0513-353X/images/top-banner3.jpg|#|菊花
https://www.ahs.ac.cn/images/0513-353X/images/top-banner4.jpg|#|灵芝
https://www.ahs.ac.cn/images/0513-353X/images/top-banner5.jpg|#|桃
https://www.ahs.ac.cn/images/0513-353X/images/top-banner6.jpg|#|黄瓜
https://www.ahs.ac.cn/images/0513-353X/images/top-banner7.jpg|#|蝴蝶兰
https://www.ahs.ac.cn/images/0513-353X/images/top-banner8.jpg|#|樱桃
https://www.ahs.ac.cn/images/0513-353X/images/top-banner9.jpg|#|观赏荷花
https://www.ahs.ac.cn/images/0513-353X/images/top-banner10.jpg|#|菊花
https://www.ahs.ac.cn/images/0513-353X/images/top-banner11.jpg|#|月季
https://www.ahs.ac.cn/images/0513-353X/images/top-banner12.jpg|#|菊花

园艺学报 ›› 2020, Vol. 47 ›› Issue (1): 111-119.doi: 10.16420/j.issn.0513-353x.2019-0130

• 研究报告 • 上一篇    下一篇

‘伏令夏橙’原位转化体系的建立及优化

谢幸男*,杨 莉*,刘 范,田 娜,车婧如,靳三鹏,张永艳**,程春振**   

  1. 福建农林大学园艺学院,园艺植物生物工程研究所,福州 350002
  • 出版日期:2020-01-25 发布日期:2020-01-25
  • 基金资助:
    福建省自然科学基金面上项目(2017J01615);福建省大学生创新训练项目(201810389094);福建省省级大学生创新创业训练项目(201910389149);福建省高原学科建设经费项目(102/71201801101);广东省科技计划项目(2016B020201006,2017A070702005);广西自然科学基金项目(2017GXNSFBA198055)

Establishment and Optimization of Valencia Sweet Orange in planta Transformation System

XIE Xingnan*,YANG Li*,LIU Fan,TIAN Na,CHE Jingru,JIN Sanpeng,ZHANG Yongyan**,and CHENG Chunzhen**   

  1. Institute of Horticultural Biotechnology,College of Horticulture,Fujian Agriculture and Forestry University,Fuzhou 350002,China
  • Online:2020-01-25 Published:2020-01-25

摘要: 以‘伏令夏橙’实生苗为材料进行原位转化体系建立及优化。首先,研究侵染部位对原位转化效率的影响:使用带有PBI121载体的农杆菌分别侵染5周龄‘伏令夏橙’实生苗上胚轴切口、顶芽切口(保留/去除真叶)2次,共培养3 d后进行抗性筛选及暗处理,7周后提取叶片DNA用于转基因植株PCR鉴定;之后,以上胚轴切口作为侵染部位进一步研究苗龄对原位转化效率的影响。研究结果表明,5周龄上胚轴切口再生率为93.10%,转化率为20.68%;保留/去除真叶后侵染顶芽时,再生率分别为81.74%和94.55%,转化率分别为19.09%和17.82%;4周龄和6周龄上胚轴切口再生率为94.59%和91.50%,转化率为25.42%和19.36%。可见利用农杆菌介导的遗传转化法原位转化4周龄‘伏令夏橙’的上胚轴切口可获得较高的转化效率。原位转化操作简单、周期短、转化率高,在甜橙遗传育种研究中具有重要的应用价值。

关键词: 橙, 原位转化, 苗龄, 外植体, 转化效率

Abstract: By using the Valencia sweet orange(Citrus sinensis‘Valencia’)seedlings as plant materials,the in planta transformation system was established and optimized. The influence of infection sites on in planta transformation efficiency was firstly investigated. Agrobacterium carrying the PBI121 vector was used to infect the epicotyl incision or apical bud incision(with/without true leaves)of the 5-week-old Valencia sweet orange seedling for 2 times. After three days’ co-culture,resistance screening and dark treatments were performed. Seven weeks later,leaf DNA of the candidate transgenic plants was isolated and was used as template for PCR detection to calculate the transformation rate. The effect of different seedling ages on the in planta transformation efficiency was further studied by using the epicotyl incision as the infection site. Results showed that the regeneration rate of 5-week-old seedling epicotyl incision was 93.10%,and the transformation rate was 20.68%. The apical bud regeneration rate for with/without true leaf was 81.74% or 94.55%,and the transformation rate was 19.09% or 17.82%. The regeneration rates of 4- and 6-week-old Valencia sweet orange seedling epicotyl incisions were 94.59% and 91.50%,respectively,and the transformation rates were 25.42% and 19.36%,respectively. By infecting the epicotyl incision of 4-week-old Valencia sweet orange seedlings,high in planta transformation rate could be obtained. The in planta transformation method is easier to be applied,less time consuming and more efficient. Thus,it is of great potential to be applied in sweet orange genetic breeding.

Key words: orange, in planta transformation, seedling age, explant, transformation efficiency

中图分类号: