In order to induce somatic embryogenesis, we often resort to the supplementation of the culture medium with the organic nitrogen supplied by L-proline (6 - 9 - 12 - 25 mM/l), glutamine (8 - 25 mM/l), glycine (0,1 mM/l) or L - asparagine (0,03 mM/l), depending on the relation between the reducer and the reduced nitrogen.
In order to observe the metabolic high tide of the reserve substances from the explant cultured in vitro and also the accumulation of the amino acids from the callus, we determined the amino acid content of the explanted mature embryos and the various embryogenic callus through the Moore and Steine method (Table 1).
Initiation of the callus was achieved on NBMCd solid medium (N6 - macroelements, Chu, 1978; B5 - microelements, Gamborg, 1968; MS - vitamins, Murashige-Skoog, 1962), added with 3.0 mg/l 2,4 D; 30 g/l saccharose; 7.0 g/l agar; pH = 5.8.
To induce somatic embryogenesis, calli were transferred onto NBMC3 solid medium (N6 - macroelements, Chu, 1978; B5 - microelements, Gamborg, 1968; MS - vitamins, Murashige-Skoog, 1962), with: 1.0 mg/l kinetin; 30 g/l saccharose; 7.0 g/l agar; pH = 5.8 for 3 weeks.
Dry weight (m) was used to assess the size and development of the callus. We observed significant modifications in the content of each amino acid.
Larger embryo mass is, correlates with a higher percentage content of amino acids:
Lc 15 (m = 20.3 mg) - 11.61 g protein
amino acids/ 100 g dry weight;
Lc 464 (m = 17.8 mg) - 10.93 g protein amino acids/ 100 g dry weight.
The amino acid content clearly diminishes at the same time as the callus size as follows:
Lc 3 (m = 21.3 mg) - 10.85 g protein
amino acids/ 100 g dry weight;
W 153R (m = 25.7 mg) - 7.31 g protein amino acids/ 100 g dry weight;
Lc 15 (m = 44.1 mg) - 6.25 g protein amino acids/ 100 g dry weight;
Lc 3 x A 188 (m = 72.2 mg) - 4.24 g protein amino acids/ 100 g dry weight.
These findings show that, for growth and differentiation of the callus, an important part of amino acids initially contained in the embryo is consumed. On the other hand, we find that all the analysed calli have lower content of proline, valine, glycine, histidine, lysine and arginine, suggesting that the transformation of these into other amino acids or compounds of a different nature (alkaloids, anthocyanins, hormones, etc). The higher the amino acid content in the explant, the better the chances of a vigorous embryogenic callus capable of supporting the regeneration ability.
On this basis we suggest that the changes in callus amino acids are a sign of metabolic processing and can be used to select callus genotypes with a higher performance in culture. It is evident that a callus obtained from the hybrid (Lc 3 x A 188) explant exhibits heterosis characteristic of the F1 generation even under conditions of in vitro culture.
Table 1. The content in amino acids
of the mature embryos and the embryogenic callus obtained from 5 genotypes
of maize (Zea mays L.) (mg/ 100 g dry weight)
|Lc 15||Lc 464||Lc 3||Lc 15||W 153 R||Lc 3 x A188|
|m = 20.3 mg||m = 17.8 mg||m = 21.3 mg||m = 44.1 mg||m = 25.7 mg||m = 72.2 mg|
|TOTAL AMINO ACIDS %||11.61||10.93||10.85||6.25||7.31||4.24|
PRO - proline; GLU - glutamic acid;
LYS - lysine; TAA - total amino acids;
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