Acquirement of leaf wrinkledness mutants from Brassica allosextuple species

Mutants are important in crop breeding and biotechnology research. Isolated microspore culture technique has been used to assist in generating mutants in some species. CGMCC No 2553 is a Brassica allosextuple species (AABBCC, n=27), which owns A, B and C chromosomes simultaneously. A simple, rapid and economic method to get leaf wrinkledness mutants from it was founded in this paper. The method mainly includes two steps. First, microspores isolated in the buds from 2.0 to 3.5 mm in length of CGMCC No 2553 were first incubated at 32.5°C for 24 h then at 25 °C for 14 days in the darkness. Cotyledon embryos were transferred to B5 solid culture medium and then recultivated for 4-5 times. Thus regeneration plants were acquired. Second, after these DH plants were set at 4-5°C for 15-20 days they were grew in greenhouse without heating apparatus at mid February in Beijing or they were directly grew in greenhouse without heating apparatus at January in Beijing at naturally lower temperature for 30-40 days. Then some DH plants could become leaf wrinkledness mutants growing some days later. Results showed 172 DH plants in 2006 and 230 DH plants in 2007 were gotten via isolated microspore culture technique. 8 leaf wrinkledness mutants were gotten from 2006 to 2007. 4 leaf wrinkledness mutants of 175 DH plants in 2007 were gotten. The highest rate of getting mutants was 2.29%. 2 mutants had gotten seeds. The leaf wrinkledness mutants mainly showed wrinkledness in the surface of preceding leaves at the seedling stage. Then the wrinkledness leaves could transfer to normal ones and some also keep the wrinkledness later with their growth. Further studies showed some DH plants could generate more than two stems in which mutant and wild type often burgeoned at the same parent. The mutants had the same characteristics as wild types except for the leaf wrinkledness, therefore they were the better materials for studying wrinkledness characteristics. They are useful in studying heredity, cloning the wrinkledness gene(s) linked to leaf mutants of Brassica allosextuple CGMCC No 2553 in the future.