II.15. Production of monoploids in barley.
C. J. Jensen. Danish Atomic Energy Commission Research Establishment Risø, DK-4000, Roskilde, Denmark.
Following the technique described by K. J. Kasha et al. (1970) we have at Risø set up a programme to produce monoploids of barley for utilization in our genetic work. The technique is seen as a very efficient way to resolve, on a gametic level, the genetic constitution of F1's, and gives completely homozygous plants immediately.
The method entails crossing the barley of agriculture, Hordeum vulgare L. (2n = 2x = 14) with the wild, diploid, cross-pollinating and perennial H. bulbosum L. (2n = 2x = 14). From about two to three week old seeds developing from these crosses the embryos are cultured in vitro. Almost all plants originating from these cultures are monoploids of barley (2n = x = 7), hybrids are very rare.
The production of monoploid barley from interspecific crosses between diploid barley (VV) and diploid H. bulbosum (BB) at Risø for the period spring-summer 1972, can be seen from the following table:
The reduction of theoretically possible embryos in the seeds obtained to the actual number of embryos cultured is mainly due to lack of embryo formation or early embryo abortion in about half of the seeds set. From 3,087 embryos cultured 139 plants were obtained, two of these were hybrids and 137 were monoploids of barley. The frequencies of monoploids obtained from embryos cultured from these interspecific crosses at Risø was about 4.5%.
Keeping the various steps in mind which make up the monoploid production procedure (e.g. growth of crossing partners, fertilization, embryo formation and culture in vitro) it is very encouraging that we can reproduce at Risø as high frequencies as reported from Guelph, Canada (K. J. Kasha et al., 1970 and personal communication, 1972).
The success with this technique to produce monoploids very much depends on: the vigor, health and growing conditions of the plant material on which the embryos are induced; the quality of the pollen and on the embryo culture technique. By concentrating on these points we hope to stabilize and improve the success rate. Using a water culture technique (a modified Hoagland solution) we handle detached shoots of barley from the time of emasculation to the harvest of the seeds. Besides easing pollination procedures (we place cut shoots of pollen releasing H. bulbosum along with barley shoots and thus obtain a constant flow of pollen on female barley florets) we find that we get slightly larger embryos on these cut shoots. By using a new synthetic embryo culture medium, Barley Medium II (K. Norstog, 1973), we obtain better and faster growth and get a higher success rate than on Gamborg's medium B5.
When embarking on our programme (November 1971) one serious drawback was the difficulty of getting suitable material of diploid H. bulbosum. In the wild state it grows in some isolated areas of Italy and southern Spain and in parts of Morocco, Tunesia and Cyrenaica. At Risø we have now a fair collection of diploid H. bulbosum from these mediterranean countries. (Colleagues finding it difficult to obtain H. bulbosum material may write to us.)
References:
Jensen, C. J. 1973. Production of monoploids in barley: A progress report - Proceedings of Eucarpia/FAO/IAEA Conference on Mutation and Polyploidy, Bari, Italy, 2-7 October, 1972 (in press).
Kasha, K. J. and Kao, K. N. 1970. High frequency haploid production in barley (Hordeum vulgare L.). - Nature 225:874-876.
Norstog, K. 1973. New synthetic medium for the culture of pre-mature barley embryos. - In vitro 9:4-6.
