II. 5 "In vitro" culture of barley.
A. Cattoir-Reynaerts and M. Jacobs. Plantengenetica. Vrije Universiteit Brussel, B - 1640 St. Genesius-Rode, Belgium. "R"
"In vitro" culture of cereals still raises several major problems. Although the introduction of 2,4-D in tissue culture methods resolved the problem of callus induction, most calluses are not actively growing and fully established callus very rarely regenerates (4,5,6,7).
In this respect barley is no exception. Although callus could be obtained from several organs, e.g. mature and immature embryos, apical and lateral shoot meristems, anthers, anther filaments and root sections, subculturing and callus regeneration are no simple problems.
First we repeated the experiments of Cheng and Smith (3), using the same cultivars Mari and Himalaya and two other cultivars, Legia and Capri. However we were unable to confirm their results. Callus was obtained from shoot tips, but the same basal medium containing only 2,4-D (2 mg/l) gave much better results than the medium of Cheng and Smith containing 2,4-D and IAA as auxins and 2iP as a cytokinin. None of the induced calluses showed the very high growth rate mentioned by the authors and transfer on medium without hormones, even of primary callus, was not followed by regeneration into whole plants. Analogous results were obtained by Koblitz et al. (4) and Wilson et al. (7).
We studied callus induction from mature embryos in detail and tried to improve environmental conditions for subculturing and regeneration. Hulled seeds were sterilized with Panogen-N (Liro Belgium) (24h, 0.1 ml/10 g seeds), naked seeds by immersion in 70% ethanol, followed by a treatment with acidified Ca-hypochlorite (5 min.). Excised embryos were resterilized for 10 min. with saturated Ca-hypochlorite. As for shoot tips, basal medium of Cheng and Smith, with 2,4-D as the only hormone gave the best results, but 5 to 10 mg/l of 2,4-D was required for optimal callus induction. Light conditions did not play an essential role. Callus was induced in the dark, in continuous light and in a cycle of 16 hours light, 8 hours dark.
This callus was subcultured in the dark, at 24°C, on the same basal medium, containing only 2 mg/l 2,4-D. On this medium the fresh weight of the callus doubled after one month. Several treatments were used to improve callus growth. None of them was successful, the highest growth rate being achieved on basal medium with 2 mg/l 2,4-D or 5-10 mg/l IAA. Higher auxin concentrations were inhibitory. Neither CPA nor NAA could be considered as appropriate auxins. Prolonged subculturing on media with IAA as the only hormone stimulated root formation. Caseine hydrolysate (0.1%) did not stimulate growth, whereas yeast and malt extracts and cytokinins (0.05-0.3 mg/l either 2iP or kinetin) were inhibitory. All these results were obtained with cvar. Capri. Afterwards thirty other varieties, including winter as well as spring cultivars were tested. Although there were some minor quantitative differences, none of them seemed more adapted to "in vitro" culture. No good growing callus was obtained directly after induction.
Meanwhile we were able to select, by repeated subculturing of the best growing parts of the embryo callus, good growing callus strains. Doubling time was about five days in liquid as well as on solid medium with 2 mg/l 2,4-D. This callus was white and rather hard. It contained several cell types, but no vascular elements. Sixty percent of the observed metaphases showed 28 chromosomes (4n). Some cells contained more than hundred chromosomes. Polynucleated cells were also observed. In liquid culture the suspension consisted of small rather uniform aggregates (20-100 cells); there were only few isolated cells.
Various regeneration media were tested, as well on primary embryo callus as on good growing strains. Hormone free medium is frequently used for regeneration of cereals. Transfer of primary barley embryo callus on this medium in a light cycle of sixteen hours light/eight hours dark caused regeneration of green rootlike structure, but no shoots or whole plants were formed. Comparable results were obtained with media containing IAA or NAA (0.01-0.05 mg/l) as an auxin and either kinetin, benzyladenin or 2iP (0.05-0.5 mg/l) as a cytokinin. Whole barley plantlets were only obtained from primary anther calluses, induced and regenerated as described by Clapham (1,2). Most of the regenerants were albinos.
On the other hand fully established, good growing calluses did not react at all to the regeneration treatments. This is not unexpected, since the isolation of fast growing callus strains took several months. The abnormal karyotype is probably also due to a rather long "in vitro" culture period. These difficulties could be avoided by a rapid and systematic isolation of fast growing strains. With this intention, the effect of the origin of the callus and of the induction conditions is studied.
The very low initial growth rates of the calluses can be explained in different ways. On the other hand it may be possible that the original primary callus contains only few healthy cells, able to divide rapidly. This should also explain the low regeneration capacity. By subculturing we select specifically for this type of cells. In order to differentiate between both hypothesis, comparative cytological and biochemical studies will be made between primary and fully established, fast growing calluses.
References:
Clapham D. Z. Pflanzenzuchtg., 69, 142-155. (1973).
Clapham D. Z. Pflanzenzuchtg., 65, 285-292. (1971).
Cheng T. Y. and H. H. Smith. Planta (Berlin) 123, 307-310. (1975)
Koblitz H. and G. Saalbach. Biochem. Physiol. Pflanzen 170, 97-102. (1976).
Mascarenhas A. F., Meera Pathak, R. R. Hendre and V. Jagannathan. Indian J. of Exp. Biol. 13, 103-107. (1975).
Mastaller V. J. and J. Holden. Plant Physiol. 45, 362-364.(1970).
Wilson H. M., B. Foroughi-Wehr, G. Mix and H. Gaul. B.G.N. 6, 86-87. (1976).