PRODUCTION OF SPONTANEOUS DIPLOID LINES FROM ISOLATED MICROSPORES FOLLOWING CRYOPRESERVATION IN SPRING RAPESEED (BRASSICA-NAPUS 6)

Cryopreservation of microspores provides a potentially powerful means for increasing percentage of spontaneous diploids in microspore-derived populations in rapeseed (Brassica napus L.). This study was to investigate the production of spontaneous diploid lines in relation to steps of cryopreserving microspores of two spring rapeseed breeding lines G-231 and M3-124. Microspores of both genotypes were isolated and subjected to cryopreservation. The step of cooling process (before liquid nitrogen immersion) in a cryopreservation protocol resulted in high percentages of spontaneous diploids in lines regenerated from frozen microspores in both genotypes. Liquid nitrogen immersion and subsequent thawing process had no influence on the percentage of spontaneous diploids. A linear relationship between percentage of spontaneous diploids and cooling temperatures was detected. The lowest cooling temperature of -40-degrees-C yielded the highest percentages of spontaneous diploids (51.1 % in G-231 and 47.7 % in M3-124, respectively), which were more than doubled in comparison to those in the corresponding unfrozen controls. The current study indicated that cold treatment may be utilized to greatly enhance the percentage of spontaneous diploids in rapeseed breeding programs.