Green haploid plants from unpollinated ovary culture in tetraploid wheat (Triticum durum Defs.)

In Triticum durum Defs., gynogenesis was investigated on six genotypes, Cocorit, Isly, Jori, Oued Zénati, Sarif, issued from Morocco,and Cham1 from Syria. The experiments described here were made during two seasons (spring and summer) in which batches of 1,036 and 3,750 unpollinated ovaries were used. A 4 ^°C pretreatment was applied, during 7, 11 or 15 days and the A (modified Jähne's et al.,1991) and B (modifed San N?um's, 1976) induction media were compared. The spikes were harvested when microspores were at bi- or trinucleate stage. After the 4 ^°C cold pretreatment, they were sterilized, and the excised ovaries underwent the following phases and sequence of successive media. The A or B ‘induction’ media, induced cell divisions in the female gametophytes, and after 4 to 9 weeks in dark conditions, the swelling ovaries burst. The growing calluses were excised and transferred to 16 h. light onto a differentiation medium (dif); there generated shoots were then placed on a development (dev), followed by a rooting medium (r). The comparison of the two experiments showed that summer was the best season, for in vitro unpollinated ovary culture; this period corresponds to a growth of mother-plants during spring, while those developing in winter gave rise to lower scores. In Experiment 1 the best level of regenerated plants per 100 ovaries were obtained for Isly with 14.3% and for Oued Zénati with 10.1%. In Experiment 2, the best values were those of Isly with 21.8%, after a 7 day cold pretreatment and use of induction medium B, and of Cocorit with 18.1%. Jori, exhibited good results, in three define situations, with 17.1%, 16%, and 9.3%.Only Cham1 and Sarif showed very low scores or no plants. Jori remaining aside, for each genotype, the best values rose up from a specific treatment combination, as a very strong interaction was expressed between genotype, induction medium and cold pretreatment duration. After this ‘first phase’ of regeneration by ovary culture 150 haploid plants were obtained. When combining these results with other attempts, a total number of 191 green plants was regenerated, 90 for Isly, 74 for Jori, 19 for Cocorit, 7 for Oued Zénati and 1 for Cham1. In a ‘second phase’, permanent regenerating callus lines were obtained through subculture on dif medium. Depending on genotype, 2 to 9 transfers (up to one year for Jori) were performed, giving 507 regenerated plants. From direct regeneration and subcultures, a total of 698 plants were produced, all green and mostly haploid. Fertile doubled haploid plants were obtained, either spontaneously or by colchicine treatment, for all these genotypes. These results are of interest for breeders, who need pure lines for genotypes evaluation and for the creation of improved homozygous varieties.