| Radish(Raphanus sativus L.,2n=2x=18),is a kind of cruciferous biennial herbs,with swelling root for edible organs,rich nutrition.In vitro regeneration technique is an important method for germplasm innovation and genetic transformation.It can cultivate a large number of plants with the same donor in a short time.The results show that radish is a kind of regenerated and recalcitrant plant,but some other plants in the Cruciferae can establish efficient regeneration system in vitro.However,there are still a lot of problems in the application of in vitro regeneration system,such as genotype,hormone concentration and proportion,application of exogenous additives.This study is based on the appropriate seed disinfection method,selected four aspects from the genotypes,hormone concentration and proportion,explant types and exogenous additives,on radish adventitious bud regeneration factors;explore the effect of culture medium NAA concentration on rooting of regenerated shoots;determination of the 'XBC'hypocotyls dedifferentiated into callus in the process of antioxidase activity and the soluble protein content change,in order to construct a radish in vitro regeneration system and genetic transformation foundation.The main results are as follows:1.Through the study of the factors influencing the regeneration of radish in vitro,we found that the genotype could affect the rate of callus formation,the rate of browning and the regeneration rate of adventitious buds.Three types of explants segments(hypocotyls,cotyledons with petiole and petiole)form callus is easy,the three callus induction rate was higher than 80%,but the formation of adventitious buds is difficult.But in the cotyledons with petiole callus was the highest(93.33%),followed by petiole,hypocotyl was the worst.Exogenous addition of silver nitrate(5 mg·L-1)and TDZ(0.7 mg·L-1)can improve the regeneration of adventitious bud regeneration coefficient,increasing the number of buds produced by the regenerated buds,and both of them can effectively reduce the browning rate,especially with the addition of silver nitrate,greatly reducing the rate of browning,the browning rate remained at around 5%.2.The rooting of regenerated shoots is easier.With the increase of NAA concentration in culture medium from 0.1?1.2 mg·L-1,the rooting rate and average rooting number showed a trend of increasing first and then decreasing.Among them,the concentration of NAA was 0.5 mg·L-1,the rooting rate(shoot rooting plants inoculated shoot regeneration/percentage of total plants)can reach 76.67%,the average rooting number per plant is the highest,reached 9.3 per plant.3.The activity changes of POD,SOD and CAT and the content of soluble protein in hypocotyl callus formation of 'XBC' were studied,for the establishment of in vitro regeneration system provides certain physiological and biochemical basis.Three kinds of culture medium(control group:no silver nitrate and TDZ;experimental group 1:5 mg·L-1 silver nitrate;experimental group 2:0.7 mg·L-1),the enzyme activity and soluble protein content of the dynamic changes.The activity of POD in the three groups was similar,and the activity increased at first and then decreased,at 10 d to 20 d,it gradually rises and remains at 90 U·min-1·g-1·FW?Among them,5 d and 10d two nodes,the experimental group 1 and 2 of the enzyme activity values were higher than the control group.The change trend of CAT of three groups were different,the CAT numerical control group has been in decline,to 15 d stable,maintained at about 5 U·min-1· g-1·FW;the experimental group 1,CAT numerical initial cultivation has been falling,until 15d gradually increased;the experimental group 2,in 0 d to 20 d in the form of wave shape change,20 d to 25 d,suddenly fell significantly.The trend of SOD activity in the three groups was also the same,after to 5 d and 15 d,both of them were on the rise,and the activity of SOD decreased from 5 d to 15 d.Among them,5 d,10 d and 15 d the three nodes,the experimental group 1 and 2,the SOD activity was higher than the control group.Three groups of soluble protein content in 5.0-9.0 mg·g-1(FW floating.Among them,the change amplitude of the experimental group 1 and 2 were greater than those of the control group,and at 25d,the relationship between the content of soluble protein:the.experimental group 2>the experimental group 1>control group.These data show that in the hypocotyl during in vitro cultured in a complex biochemical and physiological changes,experimental group 1 and 2,the change trend of enzyme activity and soluble protein was more significant,adding silver nitrate and TDZ in a certain extent can affect plant regeneration in vitro. |
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