26. Production and characterization of doubled haploids from anther culture of the F1s of Oryza sativa L. x O. glaberrima Steud.
  E.C. ENRIQUEZ 1, D.S. BRAR 2, T.L. ROSARIO 3, M. JONES 4 and G.S. KHUSH 2

1) Western Mindanao State University, Zamboanga City, Philippines
2) International Rice Research Institute, Los Banos, Philippines
3) University of the Philippines, Los Banos, Philippines
4)WARDA, Ivory Coast, Africa

The genus Oryza has two cultivated and 22 wild species with 2n=24 or 48 chromosomes. Of the two cultivated species, O. sativa (2n=24, AA) is grown worldwide whereas O. glaberrima (2n=24, AA) is grown in a limited area in West Africa. O. glaberrima is low yielding but has useful genes for resistance to rice yellow mottle virus, African gall midge, nematodes and good level of tolerance to abiotic stresses such as drought, iron and

aluminum toxicity and phosphorous deficiency. It is also an important source of weed competitiveness. We are trying to combine the high productivity of O. sativa with tolerance to abiotic stresses of O. glaberrima. The two species show strong reproductive barriers and their F1 hybrids exhibit high level of sterility (Sano et al. 1979). Several sterility genes differentiate these two species (Oka 1988). Anther culture is being explored to overcome sterility in such crosses and to produce homozygous lines, fix recombinants and use such doubled haploid (DH) lines as mapping populations.

We cultured anthers on N6 medium of 75 F1s from crosses of 10 varieties of O. sativa with 18 accessions of O. glaberrima. The calli were transferred to MS medium supplemented with 1 mg/l each of BAP, kinetin and NAA. After 3 to 4 weeks, regenerated shoots were transferred to 1/4 MS medium devoid of any auxin. After 2-3 weeks, the seedlings with well developed roots were transplanted in soil. In general, the callus induction frequency was low and ranged from 0.0-18.6%. Upon culture of 45,400 anthers from 75 F1s; we did not obtain any calli from 34 crosses. The other 41 F1s showed on an average 1.3% callus formation from 144, 160 cultured anthers.

The plant regeneration ranged from 0.0 to 77.0%. The anther derived calli from 16 F1s did not show any plant regeneration while in the remaining 25 F1s, callus induction and plant regeneration varied among genotypes (Table 1). We have produced 562 DH lines and then characterized based on plant morphology. However, we obtained 137, 138 and 65 plants respectively, from three crosses e.g. IR68552-5-3-2 x TOG 6589, IR68037-AC-24-1 x CG14. Others produced less than 30 plants each (Table 1). Among O. sativa lines, an elite breeding line of new plant type (NPT), IR68552-55-3-2 was found to respond better for producing green plants in crosses with O. glaberrima. Similarly, O. glaberrima accession, CG14 in crosses with some of O. sativa parents responded favourably to anther culture. The DH lines showed very high seed sterility, 56.2 to 100.0%.

The results indicate strong genotypic differences for anther culturability both for callus induction and plant regeneration. Furthermore, callus induction and plant regeneration from anther culture were found to be independent of each other (Table 1). As an example, in the cross IR68552-5-3-2 x TOG 6589, 6.2% anthers formed callus and showed 77.0% plant regeneration whereas in IR68703-AC-24-1 x CG14, 18.6% anthers underwent callus formation but showed only 34.4% plant regeneration. High sterility of DH lines is indicative of presence of several loci for sterility differentiating the Asian and African rice species.

References

Jones, M.P., M. Dingkuhn, G.K. Aluko and M. Semon, 1997. Interspecific Oryza sativa L. x O. glaberrima Steud. progenies in upland rice improvement. Euphytica 92: 237-246.

Oka, H.I., 1988. Origin of cultivated rice. Developments in Crop Science Vol. 14, Japan Scientific Society Press Tokyo and Elsevier, Amsterdam.

Sano, Y., Y.E. Chu and H.I. Oka, 1979. Genetic studies of speciation in cultivated rice: genetic analysis for F1 sterility between O. sativa and O. glaberrima Steud. Jpn. J. Genet. 54: 121-132.