Establishment Of Tuber Mustard In Vitro Culture System And The Study Of The Induced Polyploid By Colchicine(Brassica Juneea Var.Tumida)

In this experiment, we studied some important influencing factors in the progress of tuber mustard (Brassica juneea Czern.et Coss.var.tumida) vitro plantlet regeneration by the method of tuber mustard lines in vitro culture and seedling polyploid induction. The material used in the experiment are4tuber mustard strains named B2-1014, B2-1020, BZ(?)-1005, BZ(?)-1003respectively. We established the system of tuber mustard polyploid induction, in order to get diploid plant and did preliminary study on embryoids induction from isolated microspore culture. The results as follows:We induced successful callus result in tuber mustard in vitro culture using the shoot tips and hypocotyls of four strains. One of them that is B2-1014had the highest rate of induction.It was49.0%. We used the strain B2-1014, which had the highest rate of induction strain in vitro culture. The optimum regeneration medium were screened callus induction medium (MS+0.5mg/L6-BA+0.8mg/LNAA),Adventitiousbud induction medium(MS+2.0mg/L6-BA+0.2mg/LNAA); Adventitious root induction medium(MS+0.2mg/LNAA). Hypocotyls and shoot tips had the same optimum regeneration medium, but had different induction results. Isolated culture of shoot tips: the inductivity of the callus was95.6%, the regeneration rate of the adventitious bud was95.6%and the rooting rate was100%. Isolated culture of hypocotyls:the inductivity of the callus was up to90.0%, the regeneration rate of the adventitious bud was92.2%and the rooting rate was97.8%. There was a significant difference in the regeneration frequency in different seedling ages in vitro. The explantation was the stem apex which has the strongest differentiation capacity with the seedling age5d and that of the hypocotyls is4d.We could both get the tetraploid plants, through tissue culture and seedling inducted the polyploidy tuber mustard. And there were different conditions and different approaches may lead to different induction results. In tissue culture, when the B2-1014was used, the stem apex explants showed the best induction effects, and the best induction condition is (concentrations of colchicine0.4%, culture time48h), in which we obtained the highest doubling rate30%for tetraploid. The induction of other treatment showed that:the hypocotyls of isolated culture hypocotyls is concentrations of colchicines0.4%, culture time48h, the doubling rate of tetraploid is15.6%. Seedling induced the polyploidy of tuber mustard, then drip growing point(2.0g/L)and dip the root (40mg/L, culture time3d), simultaneous processing the highest doubling rate of tetraploid is14.4%; Dip the root, the concentrations is40mg/L, the doubling rate of tetraploid is7.8%. Polyploid of different lines of the tuber mustard had different the rate of induction. The best treatment was the lines of the tuber mustard B2-1014. It was significantly higher than other lines. Different methods had the same the optimum seeding age,3leaves of tetraploid time had the higest rate of induction.Four tuber mustards were used in the study of isolated microspore culture, from which we can know the influence of different lines of tuber mustard and different basic medium for embryoid induction. The results showed that, in different combinations of tuber mustard line and basic medium, the combination of tuber mustard line B2-1014and basic medium1/2NLN showed the best induction effect, the mutation rate was31.1%. But the mutation rate of tuber mustard lines B2-1020, BZ(?)-1005and BZ(?)-1003in basic medium1/2NLN were8.9%,5.6%and4.4%respectively.