302 EMBRYOGENESIS IN PLANTS 



When the growth of excised embryos of ten species of Datura was tested 

 in culture media in which sucrose, dextrose, levulose, mannose and 

 glycerol were used as the sources of carbon, it was found that the 

 different species varied considerably in their reactions to these com- 

 pounds. For example, D. metel grew well with mannose but poorly with 

 sucrose and the reducing sugars, and not at all with glycerol ; whereas 

 D. innoxia could not utilise mannose as well as sucrose or dextrose, and 

 could not use levulose and glycerol. In most species, sucrose is still the 

 best source of carbon thus far tested. 



LaRue (1936) succeeded in culturing small embryos, about 0-5 mm. 

 long, of species from a number of different families. Inorganic media 

 alone proved inadequate for the growth of young embryos, sugar and 

 growth-regulating substances, e.g. indoleacetic acid, yeast extract, etc., 

 being essential. 



Whereas seedlings increase in size mainly by cell enlargement, 

 embryos do so by continuing in an active state of cell division. The 

 factors controlling the growth of embryos are thus not the same as 

 those which control the enlargement of seedhngs. But the embryonic 

 regions of the latter, i.e. their apical growing points, may be affected 

 by those biochemical factors which are active in the embryo. 



Impetus was given to the study of embryo culture by van Overbeek, 

 Conklin and Blakeslee (1941, 1942) and van Overbeek (1942) when they 

 showed that whereas mature embryos of Datura stramonium are 

 completely self-sufficient in respect of growth factors, and on simple 

 culture media containing minerals and sugar can grow directly into 

 seedlings, very small excised embryos do require growth factors. Such 

 factors occur in coconut milk. In vitro cultures of embryos, only 

 slightly beyond the proembryo stage when excised, remained embryonic 

 in the presence of coconut milk for at least ten days when kept in 

 darkness. Root growth was suppressed. Coconut milk contains at 

 least three growth-regulating substances: (i) the embryo factor, which 

 makes for cell proliferation or continued meristematic activity; (ii) 

 auxin in concentrations sufficient to cause inhibition of root growth; 

 and (iii) a factor which causes an increase in the growth of the coty- 

 ledons, i.e. a leaf-growth factor, the effect of which is particularly 

 marked in the presence of light. 



When the embryo factor was added to a culture medium (containing 

 agar, minerals, dextrose, vitamins 81, 86, C, pantothenic acid, nicotinic 

 acid, adenine, glycine and succinic acid), very small embryos increased 

 up to 500 times, and even up to 3,500 times, their original volume in 

 the course of a week. The embryos were quite normal in appearance 

 but all their cells, as ascertained by various means, were apparently 

 involved in the proliferation. In the absence of the embryo factor, 



