The Exine-dehisced Microspore Embryos Have the Proper- suspensor Differentiation in Molecular Level

doi:10.11983/CBB15091

Abstract

Abstract: Because of sampling difficulty, research of the early stages of zygotic embryos is difficult. Previous studies showed that the exine-dehisced microspore embryogenesis system can stimulate the differentiation pattern of the zygotic embryo; an alternative approach is to use exine-dehisced microspore embryos as a substitute for zygotic embryos for relevant research work. However, we lack adequate evidence for this approach at the molecular level. In this study, we verified that the WOX family of genes could be markers of the differentiation of the embryo proper or suspensor in Brassica napus and showed that the embryo proper/suspensor in the exine-dehisced microspore embryo with suspensor could be differentiated at the molecular level by the expression pattern of the suspensor marker gene BnWOX8. These results provide solid evidence to use the exine-dehisced microspore embryogenesis system to study the early stages of the zygotic embryo. Meanwhile, we established the efficiency of the living laser microdissection technique, combined with RNA extraction from a few cells. We provide a reliable and applicable solution for detecting gene expression patterns in specific cells.

Haichao Wei, Yuan Liu, Xingchun Tang, Suxin Yang, Mingjun Zou, Shike Hu, Xianzhong Feng. The Exine-dehisced Microspore Embryos Have the Proper- suspensor Differentiation in Molecular Level[J]. Chinese Bulletin of Botany, 2015, 50(5): 573-582.

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URL: https://www.chinbullbotany.com/EN/10.11983/CBB15091

https://www.chinbullbotany.com/EN/Y2015/V50/I5/573

Figures/Tables 5

Figure 1 The normal embryogenesis system and the exine-dehisced microspore embryogenesis system of Brassica napus(A) The first equal division of the normal exine-intact rape microspore embryo (the arrow heads show the division plane); (B) The globular microspore embryo ruptured the exine (the arrow heads show the ruptured exine); (C) The big globular embryo without the obvious polarity differentiation; (D) The heart-stage embryo with the cotyledon primordium differentiation; (E) The first divided exine-dehisced microspore embryo (the arrow heads show the division plane); (F) The early globular exine-dehisced microspore embryo with the proper-suspensor differentiation; (G) The big globular embryo with proper-suspensor differentiation; (H) The heart-stage exine-dehisced microspore embryo with the cotyledon primordium differentiation. (Bar=20 μm)

Figure 2 The isolated zygotic embryos of Brassica napus and the proper/suspensor separated by the living microdissection system(A) The isolated early rape zygotic embryo; (B) The globular embryo; (B1) The FDA staining of Figure B; (C) The proper without the microdissected suspensor; (C1) The FDA staining of Figure C; (D) The suspensor without the microdissected proper; (D1) The FDA staining of Figure D; (E) The microdissected microspore embryo (arrow heads show the fracture); (E1) The FDA staining of Figure E. (Bar=10 μm)

Figure 3 The homologous domain comparison of the BnWOX and the WOX genes of Brassica napus and other species (Oryza sativa, Zea mays, Sorghum bicolor, Arabidopsis thaliana and Populus trichocarpa)The asterisk shows the highly conservative amino acid sites of the homodomain. The BnWOX gene family contains the same conservative domain and the amino acid sites with the WOX of other species.

Figure 4 The evolutionary analysis of BnWOX gene family of five species

Figure 5 The expression pattern of the BnWOX genes in the zygotic and microspore embryos and the proper/ suspensor(A) The expression pattern in the zygotic embryo. BnWOX2 and BnWOX9 expressed in the globular embryo and the proper, undetectable in the suspensor; BnWOX8 expressed in the globular embryo and the suspensor, undetectable in the proper. (B) The BnWOX8 expression pattern in the microspore embryos. BnWOX8 was undetectable in the microspores in the late uninucleate stage, expressed in the first divided normal microspore embryos, undetectable in the normal globular microspore embryos; expressed in the first divided exine-dehisced embryos, the globular microspore embryos with suspensor, expressed in the suspensor, undetectable in the proper of the globular embryos. GAPDH is the reference gene. EIM: Exine-intact microspores; EDM: Exine-dehisced microspore

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