Effects of methylene blue on microspore embryogenesis and plant regeneration in ornamental kale (Brassica oleracea var. acephala)

摘要

Homozygous inbreds is the most essential part in hybrids producting, while traditional breeding methods requires more than 6 years to obtain it. Isolated microspore culture (IMC) is an effective method to produce doubled haploid (DH) homozygous lines which only 2 need years to obtain homozygous inbreds. However, the high rate of death and the low frequency of embryogenesis induction still be the critical obstacles in practice application of this valuable technique. In this paper, three different treatment concentration were used to measure Methylene blue (MB) which has been added into culture medium for four genotype of kale. The results indicated that 8 nM MB at pH 5.84 can obviously promoted the rate of embryogenesis induction, While adding 10 nM MB into NLN-13 culture medium is the most optimum concentration in both genotype, more than 5-fold increased. Moreover, the addition of 8 nM and 10 nM MB significantly decreased the rate of embryos death in four genotype, especially in 'IS-F', 0.16-fold and 0.11-fold decease were showed when compared with the control, respectively. The suitable treatment concentration of MB application would increase the rates of direct plant regeneration, 10 nM is the optimum concentration in both genotype and in '1S-B', 2.29-fold increased in rate of direct plant regeneration was observed relative to the control. The percentage of doubled haploid rates in the four genotypes were up to 45%, in '1S-F', comparison of the doubled haploid percentage at different MB concentration showed only slightly changes. All of DH lines we gained in this paper are self-compatibility and showed highly uniformity. According to the measurement of the compatibility index, both genotype were prospective self-incompatible lines for hybrid breeding and shows a high uniformity, which will provide an effective way to accelerate the application of microspore in hybrid breeding.