Topic: 09: Shepherd purse embryo development observation

                                 In cleared seeds and on sections


Methods of cleared preparations of seeds as whole-mount samples are often used for quick
information about plant embryo developmental stage. Clearing mixtures allow focus to different
optical sections of the object without classic sectioning.

Nevertheless detailed study of seed and embryo structure demands using of histological techniques
with embedding the material into paraffin or resin and subsequent microtome sectioning.


A. Method of seed clearing

Material: fruits of shepherd purse (Capsella bursa-pastoris L.) fixed in Carnoy mixture (ethanol :
acetic acid 3 :1), clearing medium: saturated solution of chloral hydrate (8 parts of chloral
hydrate and 3 arts of distilled water) or  saturated solution of chloral hydrate in lactic acid

Procedure:

1.     Plant material is fixed in Carnoy mixture.

2.     Fixed objects are washed in ethanol (its concentration is the same which is used in the
fixative mixture). Fifed material could be stored in 70% ethanol for longer time. Fixation should
be omitted in case when we want to see chlorophyll in embryo cells.

3.     Isolate the young seeds from fruits in a drop of clearing medium on the microscopic slide.

4.     Cover with cover slip.

5.      Observe the preparation after interval of one hour of clearing.

Results:

Observe appropriate optical section and draw schematic picture of young embryo and its position in
the seed. Find different embryo developmental stages and document them with microphotography.

B. Paraffin sections

Fruits of shepherd purse (Capsella bursa-pastoris L.) fixed in FAA mixture (35% formaldehyde +
acetic acid + 70% ethanol            mixed in ratio 5 + 5 + 90) were dehydrated, and paraffin
embedded. Paraffin section were stained with alcian blue and nuclear fast red (Beneš and Kamínek
1973).


Literature:

1.             Beneš, K., Kamínek, M. (1973): The use of aluminium lake of nuclear fast red in
plant material successively with alcian blue. - Biologia Plantarum, 15 (4): 294 – 297.

2.             Braune W., Leman, A., Taubert H. Pflanzenanatomisches Praktikum II. 2^nd Ed. Jena:
VEB Gustav Fischer Verlag, 1982. 426 s. ISBN 261 700-36-82.

3.             Herr J. M., Jr (1971): A new clearing-squash technique for the study of ovule
development in angiosperms. – Amer. J. Bot. 58: 785–790.

4.             Lux A., Morita S., Abe J. and Ito K. (2005): An improved method for clearing and
staining free-hand sections and whole-mount samples. – Ann. Bot. 96: 989 – 996.


Picture of the cleared shepherd purse seed with torpedo embryo

Photographed in Nomarski DIC


funiculus   WB   chalazae

micropyle

  basal cell                                                       embryo
proper                       endosperm

  of the suspensor

     antipodes   suspensor                                seed coat