2. Inhibition of growth in maize embryos by canavanine and its reversal.

The natural amino acid canavanine, a structural analogue of arginine, is known to be a strong inhibitor of growth in Neurospora and certain other organisms. Experiments were conducted in 1949 to deteimine the effects of canavanine on growth of maize seedlings grown from excised embryos cultured in vitro. The results parallel and extend those observed in other organisms; i.e., canavanine indeed inhibits the growth of such embryos and this inhibition can be alleviated by the addition of appropriate amounts of arginine or certain other amino acids. These results seem to have significant implications for our understanding of the genetics and biochemistry of maize. A paper reporting these results has been submitted for publication in a botanical journal, and the following is a summary of the paper.

A number of inbred strains of maize were self‑ or cross-pollinated and the immature embryos were excised through asceptic techniques at time periods ranging from 18 to 40 days after pollination. These embryos were placed on the surface of artificial culture media contained in 18 x 150 mm. test tubes, and then were incubated at 30�C in a dark chamber either for 10 or for 15 days. The seedlings were then removed, dried at 80�C for 48 hours, and weighed.

It was found that canavanine added at the rate of 20 mg. per liter to the basic culture medium effectively inhibited growth in all lines tested. This inhibition was greatly reduced in those cultures to which arginine was added in concentrations of M/5000 or M/2500. Genetically different strains shaw variation in their sensitivity to canavanine and also in the degree of reversal of the inhibition when arginine is added. In some cases, there is indication that hybrid embryos are less sensitive than are their component inbreds and that in the hybrids reversal of the inhibition by adding arginine is more complete. Age of the embryo when excised also seems to be a factor in sensitivity.

Table 1.

Effect of amino acids on canavanine inhibition of hybrid maize embryos. Strain 38‑11 x CI 21

embryos were excised 24 days after pollination; those of CI 21 x R 20 at 21 days age.

The values represent mean dry weights in mg. of growth of seedlings in 15 days.

Each mean value is based on four replications.

Supplement	Concentration	Growth of 38‑11 x CI 21	% of control	Growth of CI 21 x R 20	% of control
None (control)		22.8 � 5.0		22.0 � 2.7
Canavanine	20 mg/l.	1.9 � 0.4	8.3	1.4 � 0.2	6.4
Canavanine + Arginine	M/5000	21.0 � 4.0	92.1	13.8 � 1.5	62.7
	M/2500	---		15.8 � 1.2	71.8
Canavanine + Lysine	M/5000	8.0 � 1.9	35.1	2.4 � 0.4	10.9
	M/2500	11.3 � 1.9	49.6	1.8 � 0.1	8.2
Canavanine + Glutamic acid	M/5000	8.8 � 1.2	38.6	10.2 � 1.7	46.4
	M/2500	17.0 � 1.8	74.6	12.9 � 1.3	58.6
Canavanine + Citrulline	M/5000	12.3 � 1.7	53.9	8.7 � 2.6	39.5
	M/2500	13.5 � 1.6	59.2	10.7 � 0.7	48.6
Canavanine + Ornithine	M/5000	10.8 � 0.8	47.4	10.5 � 1.9	47.7
	M/2500	15.4 � 1.2	67.5	13.2 � 1.9	60.0
Canavanine + Methionine	M/5000	4.8 � 0.3	21.0	‑-‑
	M/2500	5.2 � 11.2	22.8	‑‑‑

Besides arginine, the amino acids citrulline, ornithine, glutamic acid, lysine, and, to a much lesser extent, methionine are effective in counteracting canavanine inhibition of these embryos. This is shown in the accompanying table. Other amino acids tested were without appreciable effect. These included leucine, isoleucine, valine, threonine, tyrosine, proline, histidine, and glycine. The partial reversal of the inhibition by ornithine and by citrulline might lead to the interpretation that these compounds are precursors of arginine in maize, as has been shown to be the case in the mammalian liver and in Neurospora. The effectiveness of glutamic acid in relieving canavanine inhibition may be related to the fact that glutamic seems to be a precursor in the biosynthesis of arginine in certain microorganisms. The effect of lysine in relief of the inhibition parallels results obtained in Neurospora and Avena but the basis of this effect is still obscure.

Experiments were conducted to determine the effect of light, temperature, pH, and sucrose concentration of the medium on the inhibition. None of these factors significantly altered the response of 22‑day old embryos of Inbred K64 to the inhibition or to its reversal by arginine.

J. E. Wright, Jr. and Adrian M. Srb