II. 19. Biochemical mutants from the barley embryo selection system.
Simon Bright, Joseph Kueh, Ben Miflin, Biochemistry Department, Rothamsted Experimental Station, Harpenden, Herts, AL5 2JQ, U.K. "R"
Since 1976 we have been developing and using a selection system for biochemical mutants in barley with particular emphasis on amino acid metabolism. This system involves growing mature M2 barley embryos under aseptic conditions in the presence of a number of selective agents. The advantages of this system over seedling or tissue culture methods are (a) the mature embryos germinate readily to give a uniform population of plants which are fertile and cytologically normal, (b) the embryo contains little reserve protein so the young plants are forced to synthesize amino acids de novo and are thus very responsive to externally supplied inhibitors (Bright et al. 1978a, Bright et al. 1978b), (c) large numbers of mutations in M2 embryos can be induced by standard seed treatment protocols with sodium azide (Kleinhofs et al. 1978) and (d) dominant and recessive mutants can be recovered. The steps used in the screening system for each generation are outlined below:
1st Generation:
Sodium azide mutagenesis of seeds. Plants subsequently grown in the
field and combine harvested.
2nd Generation:
Mature seeds are dehusked, soaked overnight at 4°C in distilled
water and embryos with attached scutellum excised and air dried. Dry embryos
are surface sterilized and plated in petri dishes on agar-solidified medium
(modified Murashige & Skoog (1962) with no hormones) containing an
inhibitor at a concentration to allow 20% of normal leaf growth. After
growth for 7d under lights, plants with good growth are selected, measured
and transferred to soil either directly or after a time on inhibitor-free
medium. Plants are hardened in a humid propagator, transferred to the glasshouse
and grown through to the production of selfed seed.
3rd Generation:
Embryos from selfed seeds are tested on the selective medium originally
employed. Segregating or pure breeding resistant lines are identified and
grown on for crossing and biochemical analysis.
4th 5th 6th etc. Generations:
Pure breeding lines are identified and genetical and biochemical analysis
performed.
The inhibitors used, their concentrations and the numbers of embryos screened are given ln Table 1. Inhibition by lysine plus threonine is due to cumulative feedback inhibition of early enzymes in the aspartate pathway leading to starvation for methionine. Addition of methionine, or its precursors homoserine and homocysteine relieves the inhibition (Bright et al. 1978a, Bright et al. 1978b, Rognes et al. 1983). Inhibition by S(2-aminoethyl)cysteine (Aec) is caused by incorporation of this analogue into progenies in place of lysine (Bright et al. 1979a). It is a rather poor feedback regulator of aspartate kinase (Bright et al. 1978b, Davies and Miflin 1978) and its effects are relieved by more than equimolar concentrations of lysine (Bright et al. 1979b). Hydroxyproline (Hyp) inhibits some aspect of proline metabolism without being incorporated directly into protein (Cleland and Olsen, 1967). Its effects are relieved by proline (Kueh and Bright, 1981).
Table 1. Inhibitors and mutants selected.
The mutants which have been selected, their gene designations and their phenotypes are given in Table 2. Each selected plant is given an R(Rothamsted) number which is also used for the pure breeding resistant lines when they are available. Homozygous lines of the mutants R2506 and R6901 have not, so far, been obtained. This was also difficult in the case of R2501. Dwarf and semidwarf progeny have quite frequently been produced from resistant plants of R2501 (Fig. 1) but never from sensitive (wild type) segregants suggesting a closely linked second mutation or a pleiotropic effect of the Lt1a gene. As R3202 and R3004 plants are normal in vigour, appearance and fertility we favour the former possibility. No obvious changes were observed in chromosome preparations of resistant R2501 or R2506 progeny (A. Karp unpublished).
Table 2. Mutants, genes and characteristics
Figure 1. Five plants derived from the originally selected LT-resistant mutant R2501. Left pot: two stunted plants with prolific tillering and no stem elongation after 16 weeks. Right pot: a semi-stunted plant with short, thickened stems and two normal plants with normal ear development after 13 weeks.
Current work is centered on selection among the M2 of remutagenised seeds of R3004 for plants resistant to Aec. From these selections we hope to find mutants with decreased lysine-sensitivity of the remaining feedback site in lysine synthesis, dihydrodipicolinic acid synthase (Wallsgrove and Mazelis, 1981) which may accumulate lysine as well as threonine.
Seeds of all the genotypes mentioned above, with the exception of R6901 and R2506 are available for distribution. Write to: Dr. R. C. Macer, The British Association of Plant Breeders, Woolpack Chambers, Market Street, Ely, Cambs. CB7 4ND, U.K., requesting the Rothamsted mutants.
Acknowledgments:
Partial financial support from the European Community (Grant 470 to
B. J. Miflin) and NATO (Grant 277.80 to S.W.J. Bright) is acknowledged
as well as stimulating interaction with our colleagues within and outside
the Department.
References:
Bright, S.W.J., E. A. Wood and B. J. Miflin. 1978a. The effect of aspartatederived amino acids (lysine, threonine, methionine) on the growth of excised embryos of wheat and barley. Planta. 139:113-117.
Bright, S.W.J., P. R. Shewry and B. J. Miflin. 1978b. Aspartate kinase and the synthesis of aspartate-derived amino acids in wheat. Planta. 139:119-125.
Bright, S.W.J., L. C. Featherstone and B. J. Miflin. 1979a. Lysine metabolism in a barley mutant resistant to S-(2-amino ethyl) cysteine. Planta. 146:629-633.
Bright, S.W.J., P. B. Norbury and B. J. Miflin. 1979b. Isolation of a recessive barley mutant resistant to S-(2-amino ethyl)L-cystein. Theor. Appl. Genet. 55:1-4.
Bright, S.W.J., B. J. Miflin and S. E. Rognes. 1982a. Threonine accumulation in the seeds of a barley mutant with an altered aspartate kinase. Biochemical Genetics 20:229-243
Bright, S.W.J., J.S.H. Kueh, J. Franklin, S. E. Rognes and B. J. Miflin. 1982b. Two genes for threonine accumulation in barley seeds. Nature 299:278-279.
Bright, S.W.J., J.S.H. Kueh and S. E. Rognes. 1983. Lysine transport in two barley mutants with altered uptake of basic amino acids in the root. Plant Physiol. Submitted.
Cleland, R. and A. C. Olsen. 1967. Metabolism of free hydroxyproline in Avena coleoptiles. Biochemistry 6:32-36.
Davies, H. M. and B. J. Miflin. 1978. Regulatory isoenzymes of aspartate kinase and the control of lysine and threonine biosynthesis in carrot cell suspension culture. Plant Physiol. 62:536-554.
Kleinhofs, A., W. M. Owais and R. A. Nilan. 1978. Azide. Mut. Res. 55:165-195.
Kueh, J.S.H. and S.W.J. Bright. 1981. Proline accumulation in a barley mutant resistant to trans-4-hydroxy-L-proline. Planta. 153:166-171.
Kueh, J.S.H. and S.W.J. Bright. 1982. Biochemical and genetical analysis of three proline-accumulating barley mutants. P1. Sci. Lett. 27:233-241.
Murashige, T. and F. Skoog. 1962. A revised medium for rapid growth and bioassay with tobacco tissue cultures. Physiol. Plant. 15:473-497.
Rognes, S. E., S.W.J. Bright and B. J. Miflin. 1983. Feedback insensitive aspartate kinase isoenzymes in barley mutants resistant to lysine plus threonine. Planta. (In press).
Wallsgrove, R. M. and M. Mazelis. 1981. The enzymology of lysine biosynthesis in higher plants; partial purification and characterization of spinach leaf dihydrodipicolinate synthase. Phytochem. 20:2651-2655.
