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园艺学报 ›› 2021, Vol. 48 ›› Issue (8): 1605-1618.doi: 10.16420/j.issn.0513-353x.2020-0699

• 研究报告 • 上一篇    下一篇

芥蓝游离小孢子胚胎发生途径及2,4-D对成胚的影响

李智军1,2,*(), 曾晶1, 谭铭喜1, 谢景3   

  1. 1广东金作农业科技有限公司,广州 510640
    2广东省农业科学院作物研究所,广州 510640
    3韶关学院英东生物与农业学院,广东韶关 512005
  • 收稿日期:2020-09-15 修回日期:2021-03-15 出版日期:2021-08-25 发布日期:2021-09-06
  • 通讯作者: 李智军 E-mail:lizhijun04@aliyun.com
  • 基金资助:
    广州市科信局国际合作项目(201807010110);广东省省协同创新与平台环境建设专项(粤财教[2017]214号)

Embryogenesis Pathway of Chinese Kale Isolated Microspore and the Effect of 2,4-D on Embryo Formation

LI Zhijun1,2,*(), ZENG Jing1, TAN Mingxi1, XIE Jing3   

  1. 1Guangdong Golden Crops Agricultural Science & Technology Co.,Ltd.,Guangzhou 510640,China
    2Crop Research Institute,Guangdong Academy of Agricultural Sciences,Guangzhou 510640,China
    3Henry Fok College of Biology and Agriculture,Shaoguan University,Shaoguan,Guangdong 512005,China
  • Received:2020-09-15 Revised:2021-03-15 Online:2021-08-25 Published:2021-09-06
  • Contact: LI Zhijun E-mail:lizhijun04@aliyun.com

摘要:

采用改良DAPI荧光染色技术对芥蓝游离小孢子胚胎发生过程进行观察,同时比较培养基中添加2,4-二氯苯氧乙酸(2,4-D)对6个基因型芥蓝成胚的影响,并对再生植株的倍性和结实性进行鉴定。结果表明:芥蓝小孢子胚胎发生以B途径为主,但也存在A途径。小孢子第1次核非均等分裂或均等分裂形成不对称细胞即可建立极性特征,各类胚状体的形成似有其各自发育路径。2,4-D对成胚的影响因基因型而异,添加0.01 ~ 0.1 mg · L-1 2,4-D对‘绿宝’‘芊翠’和‘泽美’3个基因型芥蓝的成胚无显著影响;对于‘夏翠’,除0.1 mg · L-1 2,4-D显著降低子叶胚产量外,其他类型胚状体的产量不受影响;0.05和0.1 mg · L-1 2,4-D可分别显著增加‘顺宝’球形胚和鱼雷胚的产量,因而其总胚产量显著增加;对于成胚能力极强的‘顺宝2号’,心形胚、球形胚、其他类型胚状体及总胚产量均随着2,4-D浓度增大而显著提高,但子叶胚产量显著减少。添加2,4-D对‘顺宝2号’小孢子启动分裂无显著影响,但可维持或提高已启动分裂细胞的再分裂能力,减少中途退化或败育,从而提高胚产量。对获得150个再生株系进行倍性检测,单倍体株系占72.7%,DH系占22.7%,其他包括非整倍体和嵌合体在内的株系仅占4.6%。DH或单双倍体的嵌合体植株蕾期授粉后均能正常结实,而其他非整倍体(包括嵌合体)不结实或结实率极差。因此,可先根据植株花粉的有无、蕾期授粉后的结实性、叶形态以及生长势等判定出单倍体、DH和单倍体 + 二倍体植株,其他有花粉而不结实或结实不良的植株则可采用流式细胞仪检测其倍性。

关键词: 芥蓝, 游离小孢子, 胚胎发生途径, 2,4-D

Abstract:

Isolated microspore embryogenesis of Chinese kale was observed by an improved DAPI fluorescence staining technology and the effects of 2,4-D on the embryogenesis of six genotypes were compared. In addition,the ploidy and seed setting of regenerated plant lines obtained were identified. The results showed that B(equal division)pathway was the main pathway of microspore embryogenesis in Chinese kale and A(unequal division)pathway also occurred. Once the microspore nucleus undergoes asymmetrical division or symmetrical division accompanied by unequal division of cytoplasm at first time,its polarity can be established,and the formation of various embryoids seems to have their own developmental routes. The effect of 2,4-D on embryo yield varied with genotype. 0.01-0.1 mg · L-1 2,4-D had no significant effect on the yields of all type microspore embryos in three genotypes:‘Lubao’,‘Qiancui’and‘Zemei’. 0.1 mg · L-1 2,4-D significantly reduced the cotyledon embryo yield of ‘Xiacui’,but did not affect the yield of other type embryoids. The addition of 0.05 and 0.1 mg · L-1 2,4-D significantly increased the yields of globular and torpedo embryo of ‘Shunbao’,respectively,and therefore their total embryo yield increased significantly. For‘Shunbao 2’,which has strong ability of embryo formation,the yields of heart-shaped,globular,other type and total embryos increased significantly with the increase of 2,4-D concentration,in contrast,the yield of cotyledon embryo decreased significantly. The addition of 2,4-D had no significant effect on the microspore initiation division of‘Shunbao 2’,but could maintain or improve the re-division ability of the cells and lead to a reduction of the degradation or abortion during the process of embryogenesis,thus increasing the embryo yield. Flow Cytometry(FCM)detection of 150 regenerated plant lines revealed that 72.7% were haploid lines,22.7% were DH lines,and other lines including aneuploid and chimera only accounted for the remaining 4.6%. DH lines or haploid and diploid chimeras could set seed normally after pollination at bud stage,while other aneuploids(including chimeras)could not produce seeds or had a very low seed setting rate. In order to reduce the cost of FCM detection,haploid,DH and haploid + diploid chimera can be determined according to the presence or absence of pollen,the seed setting after bud pollination,leaf morphology and growth potential,and then the ploidy of other regenerated plants which have pollen but no seed or have poor seed can be identified by FCM.

Key words: Chinese kale, isolated microspore, embryogenesis pathway, 2,4-D

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