Factors of direct germination of microspore derived embryos of Brassica napus L

Isolated microspore culture is the main method of producing doubled rapeseed haploids and is widely used in research institutions and commercial companies. The protocol of rapeseed embryo production is well developed and efficient for many genotypes, but some issues remain due to the low regeneration frequency of plantlets from embryos. When the standard protocol is applied, regeneration of plantlets from microspore-derived embryos usually involves a callus-forming stage 51 followed by regeneration of adventitious shoots or secondary embryos, which prolong the period of plantlet regeneration and makes production of doubled haploids complicated. Identifying factors, which affect the frequency of direct embryo germination will increase the frequency of plantlet formation and reduce the period of DH plant production. In this work, we studied the effect of medium pH on the duration of plantlet regeneration from rapeseed microspore-derived embryos and effect of their low temperature treatment of +1 and +5℃ for 3, 6, 8, 9, 12 days in complete darkness on the embryo maturation and germination. Rising the pH of the nutrient medium from 5.8 to 6.1 increased the frequency of direct embryos germination up to 18% and the overall frequency of plantlet regeneration up to 76%. Culturing embryos at low temperatures effected the frequency of direct germination of embryos into plantlets. The maximum frequency of 44–53% direct embryo germination was observed when cultured at +1℃ for 6 and 9 days, when embryos were cultured at +5℃ the frequency of direct germination was 0–10%. In the control variant without cold treatment it was 16%.

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