Institut de la recherche interdisciplinaire de Toulouse

Association de la recherche interdisciplinaire



Somatic Embryogenesis and In Vivo Analysis of Iron Channels in Sunflower Embryos

X XuHan, C Brière, N Vallée, C Borin, AAM van Lammeren,  G Alibert and  A Souvré

( Cooperation with ENSAT-INP, Toulouse, France ) 


An experimental research by means of in vitro morphogenesis, in vivo labeling, cytofluorometry and confocal laser scanning microscopy 

  • An insight into in vivo ion channel distribution in relation to sunflower zygotic and somatic embryogenesis. 

  • A systematic cyto-comparison among zygotic embryogenic, somatic embryogenic, organogenic and  other plant tissues. 

Main conclusions obtained: 

  • High homology in PAA labeling among embryogenic protoderm of zygotic embryo, somatic embryo, and root young epidermis (protoderm), trichome and root cap, vs epidermis and protoderm in shoot apex. 

  • Callus and calogenesis devoids of or decrease PAA labeling respectively. 

  • The above tissues show different PAA labeling patterns, which indicates a physiologic role played by the tissues in ion exchange with environment via ion channels. 



A fluorescently labeled phenylalkylamine, DM-Bodipy PAA, was used as a probe for the in vivo detection of ion channels in embryonic and non-embryonic tissues of sunflower. Zygotic embryos, somatic embryos, callus, leaves, roots and shoots were analysed. Fluorescence intensity in the tissues was determined with cytofluorometry and confocal microscopy. DM-Bodipy PAA intensively labeled the protoderm and epidermis cells in both zygotic and somatic embryos. Callus cultures exhibited labeling on sites where somatic embryos developed. Labeling was, however, very weak in leaves, shoots and roots, except in the root cap and in the epidermis of the root. Considering that the location of phenylalkylamine binding sites is related to the distribution of ion channels in both animal and plant cells, the high intensity of labeling observed in the protoderm and epidermis of zygotic and somatic embryos as well as in protoderm, epidermis and caps of root tips, is consistent with the role these tissues may play in ion exchange with the environment. 

Key words: Helianthus annuus, embryo, epidermis, protoderm, phenylalkylamine, ion channel


DM-Bodipy PAA = (5-{3-[3-(4,4-difluoro-5,7-dimethyl-3a,4a-diaza-4-bora-indacen-3-yl)propionamido]phenethyl-N-methylamino}-2-isopropyl-2-(3,4,5-trimethoxyphenyl)-valeronitrile) (Molecular Probes, USA)


Fig.1:  Schematic representation of tissue preparation for Helianthus annuus embryos and probe loading control. The intensity of fluorescence was measured in transverse sections in an observation chamber. Three embryonal regions were analysed: i) the protoderm or epidermis, ii) the inner tissues, and iii) the inner tissues next to the longitudinal/radial cut face that could improve accessibility for the probe.


Figure from Protoplasma 210:52-58, 1999



Fig. 2: Determination of fluorescence intensities of DB-Bodipy PAA labeling in protoderm, inner tissue and cut face of transverse sections of zygotic embryos of Helianthus annuus. (A) Fluorescence intensities were measured after DM-Bodipy PAA labeling (i), after DM-Bodipy PAA labeling plus (-)-Bepridil post-treatment (ii), and the autofluorescence of the tissues was determined (control). (B) Specific labeling by DM-Bodipy PAA in the three tissue regions was deduced by subtraction the values (ii) of the values (i). Data represent the mean with SE of  80 sections.

Figure from Protoplasma 210:52-58, 1999 




Fig. 3a-i: Series of optical sections of an intact zygotic embryo of sunflower at the globular stage labeled with DM-Bodipy PAA and observed by CLSM. Bar = 100 mm. 

Fig. 4a-i: Series of optical sections of an intact zygotic embryo at the heart stage labeled with DM-Bodipy PAA and observed by CLSM. Bar = 200 mm. 

Fig. 5: Series of optical sections of an intact root tip of a zygotic embryo at the globular stage labeled with DM-Bodipy PAA and observed by CLSM from outer region (a & b) towards the median sections (c-e). Bar = 1 mm. 

Fig 6: Immature zygotic embryo in culture for 8 days. The lower cotyledon (lc), which is in contact with the culture medium, developed callus (c); the embryo axis (a) and the upper cotyledon (uc) developed normally. Bar = 1mm. 

Fig. 7: a) Confocal optical section of an epidermal strip in culture labled by DM-Bodipy PAA. b) Thin section of similar tissue showing embryo initiation site (em), callus (c) and epidermis (ep). Bar = 100 mm. 

Fig. 8: a) Somatic embryo (em) and its attached callus (c) observed by CLSM after DM-Bodipy PAA labeling. b-c) Another somatic embryo observed by light microscopy. Bar = 20 mm. 

Figure from Protoplasma 210:52-58, 1999 



Fig. 9: Schematic representation of DM-Bodipy PAA labeling (red shadow) in embryos and plants of sunflower during the development of zygotic embryos to plants (thick red arrows), during the culture of zygotic embryos (thick blue arrow) and during the culture of hypocotyl strips giving rise to somatic embryos (think black arrow). 


Refer to  Protoplasma 210:52-58, 1999  





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