|Title:||2,4-D對高氏柴胡葉片癒合組織誘導、增殖與柴含量之影響||Other Titles:||Influence of 2,4-dichlorophenoxyacetic Acid on Leaf Callus Induction, Proliferation and Saikosaponin Formation of in vitro Bupleurum kaoi Liu, Chao et Chuang||Authors:||陳威臣
|Keywords:||高氏柴胡;藥用植物;癒合組織;柴胡皂苷;Bupleurum kaoi;Medicinal plant;Callus proliferation;Saikosaponin||Issue Date:||Mar-2005||Publisher:||台灣農藝學會||Journal Volume:||2||Journal Issue:||1||Start page/Pages:||39-49||Source:||作物、環境與生物資訊||Abstract:||
本研究利用臺灣原生藥用植物-高氏柴胡 (Bupleurum kaoi) 組培苗葉片片段，培養於添M 0.5-4 mg L-1 2,4- dichlorophenoxy- acetic acid (2,4-D)之 1/2 Murashige and Skoog (MS) 培養基均可誘導癒合組織形成，其中以 4 mg L-1 2,4-D 處理之誘導率達 75%最佳。選擇生長快速、淡黃色且質地綿 細之癒合組織進行試驗後發現，較低濃度 2,4-D (0.1-0.2 mg L-1 )較適於癒合組織之生長與增殖，提高 2,4-D 濃度 (0.4-0.8 mg L -1 2,4-D)則降低癒合組織之鮮重與乾重。葉 片癒合組織培養於含有 0.2 mg L-1 2,4-D 之1/2 MS 培養基，結果顯示其鮮重在培養後第 1 至第 10 週呈直線上升，而後鮮重增M 減 緩，此時已達最大乾重，且外觀部分呈現褐化現象，因此繼代時間應早於培養後 10週為宜。高壓液相層析 (high performance liquid chromatography HPLC)分析結果顯示，柴胡皂苷 a、c、d (saikosaponin a, c, d; SSa, SSc, SSd) 的累積與癒合組織生長週期有密切關係，培養中期(第 4 週與第 6 週 )隨著癒合組織大量增殖，三種柴胡皂苷總合含量(SSa+SSc+SSd)濃度較 低，培養 8 至 10週之細胞成熟時 SSa+SSc+SSd 累積達最大量。惟培養後期 (第 12 週)細胞老化時，其 SSa+SSc+SSd 又再度下降。高氏柴胡葉片癒合組織經培養於含有 0.2 mg L-1 2,4-D 之1/2 MS 培養基 8 週 後，其SSa+SSc+SSd 為-1 1.12 mg g dry wt，單位產量雖然較柴胡市場品、田間高氏柴胡植株地上部與根部之含量低，但由於癒合組織具有生長週期短且 週年生產不受環境影響之諸多優 點，未來更 可藉由調整各項因子以提高細胞生長與柴胡皂苷含量，達到產業化應用之目標。
Effect of 2,4-dichlorophenoxyacetic acid (2,4-D) on leaf callus induction, proliferation and formation of saikosaponin along with callus culturing of Bupleurum kaoi, a native medicinal herb of Taiwan, have been investigated. A seventy-five percent primary callus induction rate was obtained from the explants cultured on half-strength Murashige and Skoog (MS) medium containing 4 mg L-1 2,4-D for 8 weeks in darkness. Among the varied 2,4-D concentrations (0 to 0.8 mg L-1) tested for callus proliferation, the highest callus weight was observed on half-strength MS medium containing lower concentration (0.1 to 0.2 mg L-1) of 2,4-D and increasing of 2,4-D concentration did not further improve callus proliferation. In addition, time course of callus growth and saikosaponins content were also established. There was linearly increasing in callus fresh weight from week 1 to 10 before a slowly growth occurred from week 10 to 12. The highest biomass of callus was obtained at week 10, where callus browning started. Therefore, the period for a proper growth of callus on a proliferation medium should be no longer than 10-week-incubation. Saikosaponin a, c and d (SSa, SSc, SSd) contents of callus were analyzed at two-week intervals in a total of 12 weeks span using a high performance liquid chromatography (HPLC). The total amount of SSa, SSc, and SSd (SSa+SSc+SSd), 1.12 mg g-1 dry wt, was founded reaching the peak at interval from week 8 to week 10 and diminished hereafter. The contents of SSa, SSc, and SSd in various sources of plant materials including the aerial parts and roots of eighteen-month-old field-grown plants of B. kaoi and market crude drug (Radix Bupleuri - roots of B. Chinense), were also determined by HPLC. Although the total amount of SSa, SSc, and SSd of the cultured callus was lower than other sources of tested samples, it is thought that by modifying several important factors for callus culture of B. kaoi could make this system more efficient. Cell suspension culture in a long run has a great potential and advantage to produce secondary metabolites stably year round comparing with the field-growth plants for pharmaceutical utilization.
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