Profound improvements of isolated microspores culture techniques in winter Brassica napus L.
We studied the effects of sampling stages, physical conditions (like temperature), culture conditions, embryo long-distance transportation methodology and plantlet regeneration on isolated microspores from donor plants in field. Results indicated that if microspores were sampled in bud stage instead of blooming stage to enhance the duration of microspore culture by about 9 days, embryo yield per bud reached maximum (29.61) when environmental temperature was more than 20°C. Compared with previous procedure of generating sampling buds by cutting the inflorescences, re-harvesting buds from donor plants could increase the usage efficiency of donor plants by 9 times. Use of 13% sugar solution for extracting microspores instead of B5 solution containing 13% sugar showed the similar embryo yield per bud. This improved protocol could decrease the costs of microspore culture. Compared with normal incubating method, the improved incubating method roughly resulted in the same number of embryo and appeared markedly 4 days earlier. Liquid B5 medium had several advantages in transporting embryos for long distance notably the number of embryo transported was about 8 times greater and the transported volume decreased by 7 times when compared with embryos transported in solid B5 medium. Therefore, the improved procedures could undoubtedly enhance the efficiency of microspore culture in Brassica napus.
Key words: Brassica napus, microspore culture, embryogenesis, technique improvement.