Studies On Regeneration System Of Autotetraploid Non-Heading Chinese Cabbage

The vegetable of Brassica campestris was hard to regeneration in vitro, while gene engineer and other high technology breeding techniques were based on the foundation of high frequency of regeneration system. Studies on non-heading Chinese cabbage, especially autotetraploid non-heading Chinese cabbage, which wasn't any report heretofore, had important meanings of theoretics and practice.The paper studied the regeneration system of autotetraploid non-heading Chinese cabbage(Brassica campestris ssp.chinensis). The effects of genotypes on adventitious shoots regeneration from cotyledons of six autotetraploid and two diploid non-heading Chinese cabbage were studied in vitro; The optional mediums of adventitious shoots regeneration of all materials were made by orthogonal design; The relativity between regeneration capacity and the content of endogenous hormones of cotyledons was studied. And the effects of explant types, the seedling age, agar concentration and different combinations of auxins and cytokinins on shoots regeneration were investigated in autotetraploid Cytoplasmic Male Sterility(4xCMS). Then the cotyledons of 2xCMS and 4xCMS were studied on physiological and biochemical mechanism of adventitious shoots regeneration in vitro. Finally rooting of adventitious shoots of diploid and autotetraploid materials, forming regeneration plants and acclimating these plantlets were investigated. Observing the time of bolting and flowering and identified by appearance and chromsome number. The aim of the paper was establishment of stability and high frequency of autotetraploid non-heading Chinese cabbage regeneration system. The results were as follows.Genotype was the key factor to adventitious shoots regeneration. The cotyledons were in vitro induced from six autotetraploid non-heading Chinese cabbage by orthogonal design of three factors and three levels on TDZ(0.1 0.5 and 1.0 mgL^-1), NAA(0.1, 0.5 and 0.8mg L^-1)and AgNO₃ (0 5.0 and 10.0 mgL^-1), which all could regenerate adventitious shoots. The best levels of all factors, the sequence of influencing factors and the optionalregeneration mediums of all materials were made by analysis of orthogonal design. The sequences of regeneration capacity of different autotetraploid materials were arranged according to the frequency of adventitious shoots regeneration under a same medium and the highest frequency of adventitious shoots induction . The results showed that there was no significant relativity between the content of endogenous hormones (ZRs and IAA) and regeneration capacity, While the relativity coefficient were 0.9482 and 0.8857 between the regeneration capacity and the sequence the ratio of ZRs / IAA. Comparing two groups' regeneration capacity of diploid and autotetraploid materials showed that the autotetraploid material and its diploid were all stronger on regeneration capacity. The content of endogenous hormones ZR_S and IAA was tested, and finding that the content of endogenous hormones of autotetraploid was higher than that of their diploid.Among three kinds of explants (cotyledon, hypocotyl and root) from 4xCMS, the sequence of regeneration capacity was cotyledon > hypocotyl > root. Cotyledon as explant, seedling age 5 was the best and 0.8% agar to the medium MS+O.SmgL^-1 NAA + 0.5 mgL^-1 TDZ +10.0mgL^-1 AgNO₃, the adventitious shoots regeneration frequency was 83.3%. Among three cytokinins (TDZ, 6-BA and KT) and two auxins (NAA and 2,4-D), TDZ and NAA matched could increase the regeneration frequency. The quality of regenerated plants was the better.This paper reported the changes of soluble protein content, peroxidase (POD), superoxidase (SOD) and catalase (CAT) activities and esterase isozymes ( EST) electrical phoresis during cotyledon of 2xCMS and 4xCMS cultured on the medium MS+0.5mgL^-1 TDZ+0.5mgL^-1NAA+10.0mgL^-1AgNO3+0.8% agar+3% sugar. Except SOD tended to increase, soluble protein content, POD and CAT activities tended to decline before adventitious shoot induction. All isozyme activities decrease during the phase of adventitious shoots inductio...