The Research On Callus Induction And Subculture Of Scutellaria Barbata D.Don

In this study, the stems of Scutellaria barbata D. Don plants were used as the plant materials. Firstly, the different light conditions were designed, then the effect of auxin, cytokinin, gibberellin and spermine on callus induction and subculture, and the the total flavonoids content were also studied preliminarily. The results are as follows:(1) The effect on callus induction and growth under different light conditions (darkness for four weeks, light cycle two weeks alternates darkness and 16 h/8 h light for four weekes) were explored, and different concentration of 6-BA (0.0025,0.0045 and 0.0090 mmol/L) and NAA (0.0025,0.0055 and 0.0080 mmol/L) were added in MS medium. The results showed:The callus induction rate was 0 and the highest total flavonoids content was 0.107% when explants were cultured on the MS medium. The callus induction rate was 96.67% when explants were cultured on the medium supplemented with 0.0090 mmol/L 6-BA and 0.0080 mmol/L NAA under the condition of 16 h/8 h light for four weeks, the total flavonoids content reached 3.064%, which was 28.6 times than the control group. In addition, the callus assumed the yellow-green colour, had a soft and water texture and grew well without the appearance of browning. So the best combination of the media for subculture is NAA(0.0090 mmol/L)and 6-BA(0.0080 mmol/L).(2) The effect on callus induction and growth under different combination treatment with auxin and cytokinin were explored. The results were showed: ①The combination of 2,4-D and 6-BA:The callus induction rate was 79.17% and the highest total flavonoids content was 3.096% when explants were cultured on the medium supplemented with 0.0070 mmol/L 2,4-D and 0.0025 mmol/L 6-BA. The callus induction rate was 0 and the highest total flavonoids content was 0.194% when explants were cultured on the MS medium. ②The combination of IBA and 6-BA:The callus induction rate was 41.67% and the highest total flavonoids content was 2.639% when explants were cultured on the medium supplemented with 0.0025 mmol/L IBA and 0 mmol/L 6-BA. The callus induction rate was 0 and the highest total flavonoids content was 1.367% when explants were cultured on the MS medium.③The combination of IBA and KT:The callus induction rate of all groups was 79.17%,and the highest total flavonoids content was 3.096% when explants were cultured on the medium supplemented with 0 mmol/L IBA and 0.0045 mmol/L KT. The callus induction rate was 0 and the highest total flavonoids content was 2.212% when explants were cultured on the MS medium. ④The combination of IBA and TDZ:The callus induction rate was 70.83% and the highest total flavonoids content was 3.056% when explants were cultured on the medium supplemented with 0 mmol/L IBA and 0.0045 mmol/L TDZ. The callus induction rate was 0 and the highest total flavonoids content was 0.827% when explants were cultured on the MS medium. ⑤The combination of 2,4-D and KT:The callus induction rate was 41.67% and the highest total flavonoids content was 3.070% when explants were cultured on the medium supplemented with 0.0025 mmol/L 2,4-D and 0 mmol/L KT. The callus induction rate was 0 and the highest total flavonoids content was 0.925% when explants were cultured on the MS medium. ⑥ The combination of 2,4-D and TDZ:The callus induction rate was 91.67% and the highest total flavonoids content was 2.931% when explants were cultured on the medium supplemented with 0.0065 mmol/L 2,4-D and 0.0065 mmol/L TDZ. The callus induction rate was 0 and the highest total flavonoids content was 0.410% when explants were cultured on the MS medium. ⑦The combination of NAA and TDZ:The callus induction rate was 95.83% and the highest total flavonoids content was 3.402% when explants were cultured on the medium supplemented with 0.0080 mmol/L NAA and 0.0025 mmol/L TDZ. The callus induction rate was 0 and the highest total flavonoids content was 1.275% when explants were cultured on the MS medium. ⑧The combination of NAA and KT: The callus induction rate was 41.67% and the highest total flavonoids content was 2.873% when explants were cultured on the medium supplemented with 0.0080 mmol/L NAA and 0.0025 mmol/L KT. The callus induction rate was 0 and the highest total flavonoids content was 1.876% when explants were cultured on the MS medium.(3) The effect on callus induction and growth of different concentration treatment with gibberellin and Spermine were explored. The results were showed:The callus induction rate was 100.00% and the highest total flavonoids content was 3.332% when explants were cultured on the medium supplemented with 0.005 mmol/L gibberellin. Spermine had no significant influence on the callus induction. We obtain the maximum callus induction rate (93.33%) treated with 0.5 mmol/L spermine. And the highest total flavonoids content was 3.086%, higher than other groups.(4) The flavonoids content in callus of the diploid and the tetraploid Scutellaria barbata D. Don induced by colchicine were compared. The callus induction rate of two kinds of Scutellaria barbata D. Don were 96.67% and 97.92% respectively when explants are cultured on the medium supplemented with NAA and 6-BA. The highest total flavonoids content was 3.402% when the diploid explants were cultured on the medium supplemented with NAA and TDZ. The highest total flavonoids content was 5.622%when the tetraploid explants were cultured on the medium supplemented with IBA and TDZ. The results showed that flavonoids content of the tetraploid in callus generally higher than that of the diploid.(5) The effect on callus subculture of different concentration treatment combination of three hormones(NAA,6-BA and TDZ) were explored by using orthogonal test. The optimal treatment combination was 0.0055 mmol/L NAA,0.0090 mmol/L 6-BA and 0.0025 mmol/L TDZ. The maximum growth capacity of callus was 1.544 g.