Triticale (X Triticosecale Wittmack) in vitro androgenesis in isolated microspore culture

Triticale (X Triticosecale Wittmack ex. A. Camus) microspore culture experiments were carried out to study the induction conditions required for triticale androgenesis. This is the first description in the literature of the sporophytic development of triticale microspores in isolated microspore culture. In the experiments, five winter type hexaploid (AABBRR) triticale entries were involved: one variety and four crossing combinations. The micropores were mechanically isolated in 0.3 M mannitol using a microblender to avoid the laborious process of anther isolation. A density gradient centrifuge was used to separate viable and unviable microspores, the viable ones being collected from the maltose/mannitol gradient interphase. Microspore sporophytic development was successfully induced in modified 190-2 medium supplemented with one of two growth regulators (190-D/K and 190-PAA) or without hormones (190-0). The highest induction of microspore embryogenesis was obtained using the hormone-free medium (190-0). This indicates that triticale in dba microspore development does not require an exogenous hormone signal for cell division in microspore culture. In conclusion, adventitious embryogenesis was observed during the in vitro development of triticale microspores. Albinism remained a serious problem in triticale microspore culture which was reflected by more than 50% of the regenerants being albino. In total, 126 green plantlets were transplanted into the soil and grown in the greenhouse. In a cytological study 90% of the transplanted regenerants were found to be haploid. In order to differentiate between haploid, mixo- and spontaneous diploid derivatives, an indirect ploidy evaluation method (stomatal guard cell length measurement) was successfully used. In the case of 56 plants, the stomatal guard cell length ploid level estimation was confirmed by root tip cytology.