Herbicide resistance as a marker in screening for maternal haploids
-- H. H. Geiger, S. R. Roux and S. Deimling
In crosses with inbred line Stock 6 as pollinator parent, Coe (Am. Nat. 93:381-382, 1959) observed 2.3% maternal haploids. Recently, Lashermes and Beckert (TAG 76:405-410, 1988) were able to increase the haploid frequency to 2 - 5 % using inbred WS 14 (derived from a cross between W23ig and Stock 6) as the inducer line. This phenomenon could be used as a simple, fast, and inexpensive means of haploid production if a genetic marker existed which would allow efficient screening for haploids among the regular sexual diploids.
In our experiments we investigated the usefulness of the transgenic resistance against the herbicide BASTA as a physiological marker. The resistance is inherited as a monogenic dominant trait. Resistant and sensitive genotypes can be distinguished in young seedlings by applying BASTA in a concentration of 1% to the terminal half of one leaf. Three to four days later the herbicide damage becomes visible on the sensitive seedlings whereas the resistant ones remain unaffected.
A BASTA-resistant line (kindly provided by Dr. G. Donn, Hoechst AG, Frankfurt/M.) was crossed to the inducer line WS 14 (kindly provided by Dr. M. Beckert, INRA, Clermont Fd., France), backcrossed three times to WS 14, and subsequently selfed using resistant plants for backcrossing and selfing. From the resulting BC3S1 versions of WS 14, 6 homozygous resistant plants were used as pollinator parents in the present study. Homozygosity of the resistance gene was determined a posteriori in the second selfing generation (BC3S2).
To check the effectiveness of the BASTA marker, we used an S2 line with the monogenic recessive mutant liguleless as female parent. Maternal haploids, as well as spontaneously doubled maternal haploids, should be both sensitive to BASTA and liguleless, whereas sexual (F1) seedlings should be heterozygous at the two loci and thus display BASTA resistance and normal leaf morphology. In four progenies consisting of 111, 179, 202, and 259 seedlings, the frequency of maternal haploids (or doubled haploids) was 1.0, 1.1, 1.6, and 3.8%, respectively. In all cases the BASTA sensitive plants were liguleless and the resistant ones were normal.
These results clearly demonstrate the usefulness of BASTA resistance as a foolproof marker system to identify maternal haploids. In comparison to the R-nj-embryo marker (Greenblatt and Bock, J. Hered. 58:9-13, 1967), BASTA resistance has the advantage of unambiguity and independence of the genetic background of the female parent. However, using a transgenic inducer genotype for haploid production on a commercial scale would require field experiments, for which a permit might be difficult to obtain in certain countries. The final doubled haploid lines, on the other hand, could be grown without any such restrictions.
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