| 摘要註: |
本論文建立高氏柴胡癒傷組織誘導系統，並以不同種類及濃度之植物生長調節劑，將所誘導之癒傷組織進行試管內形態發生。種子萌芽試驗結果顯示，以1.25%之次氯酸鈉處理二十分鐘的萌芽率最高（60%）。切取萌芽八週的實生苗之根及葉作為培植體，以不同濃度之生長素（2,4-Dichlorophenoxyacetic acid, 2,4-D或1-Naphthaleneacetic acid,NAA）與細胞分裂素（Thidiazuron, TDZ或N6-enzylaminopurine, BA）搭配進行癒傷組織誘導，共獲得十七個品系癒傷組織，這些癒傷組織穩定地生長並以相同的培養條件下持續進行繼代。各品系癒傷組織之形態結構有所差異。藉由增殖率試驗及癒傷組織形態分析，挑選形態為黃色或淡黃色之結構緊密且增殖率高的癒傷組織，包含BKL-7L、BKL-8R、BKL-10R、BKL-13L、BKL-13R、BKLC-7R、BKLC-12R 及BKLC-13L共八品系，繼續進行形態發生試驗。形態發生試驗過程中，各品系癒傷組織培養在光照環境，開始由淡黃色或黃色逐漸轉為綠色。其中BKL-8R、BKL-10R、BKL13R、BKLC-7R、BKLC-12R 及BKLC-13R癒傷組織在光照培養下，逐漸形成根器官。以不同濃度BA及NAA誘導下，BKL-10R、BKL-13L、BKL-13R、BKLC-7R、BKLC-12R及BKLC-13R癒傷組織產生根器官。BKL-13L 癒傷組織於0.1 mg/L BA+10 mg/LNAA、BKLC-7R癒傷組織於1 mg/L BA+1 mg/L NAA 及BKLC-12R癒傷組織於0.1 mg/L BA+10 mg/L NAA、1 mg/L BA+1 mg/L NAA試驗組中發根率為最高，比率達到100%。由NAA與BA所誘導之癒傷組織品系，在癒傷組織培養時期即有根生成，而各品系癒傷組織搭配不同濃度之BA與NAA進行形態發生誘導過程中，陸續也出現發根現象。推測NAA與BA對高氏柴胡癒傷組織具有促進發根的作用。在TDZ誘導癒傷組織形態發生過程中，BKL-7L、BKL-7R、BKL-12L、BKL-12R、BKLC-2R、BKLC-7R 及BKLC-12R共七品系癒傷組織，除BKLC-2R品系外，其餘品系在光照培養下開始轉綠，但經八週培養後，仍無發根現象。高氏柴胡細胞懸浮培養試驗結果顯示，BKL-13R品系癒傷組織細胞之平均月增殖量無論在不添加水晶洋菜之原培養基或D2培養基中都比BKL-8R品系高。現階段試驗結果顯示，BKL-13R品系癒傷組織應較有利於未來高氏柴胡進行細胞懸浮培養、基因轉殖及多倍體試驗等研究。 This thesis attempts to develop a protocol for induction of callus from explants taken from seedlings of Bupleurum kaoi, and subsequently the in vitro morphogenesis of callus that was induced by different types and concentrations of plant growth regulators (PGRs) were evaluated. It was found that the treatment of 1.25% sodium hypochlorite for 20 min gave the highest germination rate (60%). Segments taken from roots and leaves of 8-week-old seedling were used as explants for callus induction. The results displayed that there were 17 callus lines could be obtained on culture media supplemented with different combinations of auxins (2,4-Dichlorophenoxyacetic acid, 2,4-D or 1-Naphthaleneacetic acid, NAA) with cytokinins (Thidiazuron, TDZ or N6-Benzylaminopurine, BA). These callus showed stable growth and could be subcultured on the same culture medium. They possessed obvious variations on their color and shape among these callus lines. By systematically selection on proliferation rate and callus morphology, yellow or whitish-yellow compact callus, comprising lines BKL-7L, BKL-8R, BKL-10R, BKL-13L, BKL-13R, BKLC-7R, BKLC-12R and BKLC-13L, having high proliferation rate were further selected for in vitro morphogenesis. Then, all of the callus which were cultured in light for induction gradually turned from yellow or whitish-yellow into green. Furthermore, root formations were found in callus lines BKL-8R, of BKL-10R, BKL13R,-7R BKLC, BKLC-12R and BKLC-13R. Additionally, it was found that roots could be induced from callus lines BKL-10R, BKL-13L, BKL-13R, BKLC-7R BKLC-12R and BKLC-13R on media supplemented with different combinations of BA and NAA. The results showed that line BKL-13L at 0.1 mg/L BA+10 mg/L NAA, BKLC-7R at 1 mg/L BA+1 mg/L NAA and BKLC-12R at 0.1 mg/L BA+10 mg/L NAA or at 1 mg/L BA+1 mg/L NAA had a 100% rate of root formation. Callus induced from different combinations of NAA and BA had ability to form roots during callus subculture, and more roots could be obtained when they were transferred onto different combinations of BA and NAA for induction. It was suggested that combinations of NAA and BA could promote root formation from callus cultures in Bupleurum kaoi. Seven callus lines, including BKL-7L, BKL-7R, BKL-12L, BKL-12R, BKLC-2R, BKLC-7R and BKLC-12R, were used to evaluate the ability of induction in vitro morphogenesis under different concentrations of TDZ. After eight weeks, except for line BKLC-2R, all of the callus lines which was cultured in light turned into green. Besides, no roots could be found in all of the treatments. The result showed that cell suspension culture was established from callus of Bupleurum kaoi, the average of monthly proliferation of callus lines BKL-13R in terms of using liquid media by omitting of gelling agent or D2 medium were higher than BKL-8R. The results suggested that callus line BKL-13R should be more conducive to cell suspension culture, transgenic and polyploid research of Bupleurum kaoi in the future. |