臺灣博碩士論文加值系統

流蘇懸浮細胞生長於含1MG/L 2,4-D之MS基本培養基？，其生長發育過程並非同步化的進行，且呈現有優勢族群及隨生長變遷的現象。混合生長型之細胞，依優勢族群生長細胞型態可區分成：游離單細胞至多胞期、團胞期、原球狀期、球狀期、增大期、變形期、重組期及釋放期等八個發育期。並可依其生長型態及特性，將其歸納為分裂旺盛的增生相，具原表皮構造的球體相，及經逆分化作用增殖與釋放細胞或細胞群的生殖相等三個生長相。懸浮細胞不同發育相的生長速率，依細胞沈積體估算，以原球狀期者及球體相者較高，一繼代(10天間隔)之細胞增生量各為0.38 x 10及0.34 x 10。
流蘇懸浮細胞之生長周期，可以區分為二種模式或三種途徑進行循環生長。生長週期呈2至5個月的變化，其因細胞胚性潛力、細胞生殖模式及循環生長途徑等因素之而異。細胞初始培植密度、繼代培養基更新比例及繼代頻率等因子，會影響流蘇懸浮細胞培養之生長發育。流蘇懸浮細胞維持50 ml培養液含有2 ~ 3 ml細胞量、培養基以舊液：新液為1：1 ~ 1：4的比例更新，每十天繼代一次的頻率，皆有助於長期維持細胞較佳的生長勢。而pH值3.2 ~ 6.2範圍之培養基，對流蘇懸浮細胞的繼代生長並無明顯之影響；Auxin-free培養基，則影響培養之細胞族群的生長型態及循環生長週期。
流蘇懸浮細胞胚性潛力檢定，以原球狀期及球狀期之細胞團接種於再生培養基其組成為含0.4 mg/l NA、45 g/l sucrose及2.5g/l gelrite，可獲得快速大量的體胚。平板培養後起始接種體，先細胞分裂，形成明顯可見的黃色細胞團，再進一步進行體胚發生。流蘇體胚發生的形態發育步驟為原胚期、球狀胚期、心臟胚期、魚雷胚期、子葉胚期。
流蘇懸浮細胞生長發育期之胞外pH值，呈現高pH調變，其，增生相為5.3±0.3，球體相為5.6±0.3，生殖相初期為5.35±0.3，後期隨釋放途徑不同而異，循環生長途徑二會自調至5.1，循環生長途徑三則會自調至6.1。呈現高pH調變，其變域範圍為在4.7 ~ 6.8。正常的細胞系在長期繼代培養下，其pH值大多會維持在5.0以上。劣變的細胞系，其pH值在長期繼代培養後，會逐漸酸化至4.2左右。推測pH 4.7 ~ 5.0為流蘇細胞活力的緩衝區，細胞存活則胞外pH值會回復至5.0以上，劣變死亡的胞外pH值會持續低於4.7。
經流蘇胚性懸浮細胞繼代培養世代胞外pH之變化模式，可將其區分為快速反應期及自調反應期二階段，快速反應期為繼代後1 ~ 2小時內pH值快速上升，並在30小時內下降，接自調反應期，則呈現緩慢調昇pH值。進行繼代培養可衝擊及維持懸浮細胞重複胞外pH如前之變化模式，而維持胚性循環生長。若細胞失去胚性或發生劣變，繼代培養後則無此急速上升下降波動變化及自調現象。2,4-D會影響快速反應期的發生與否，但自調反應期則非由2,4-D所控制。另外，以一般pH meter測量流蘇懸浮細胞之胞外pH值變化趨向，亦會隨細胞接種量之增加而提高的趨勢，但不受繼代培養基初始調控pH位階之影響。

The embryogenic suspension cells of fringe tree (Chionanthus retusus Lindl.) was amprised of heterogenous population. According predominant cell population, which could be discriminated into eight developmental stages, including free cells and cell cluster, cell mass, pro-globular body, globular body, enlarged body, deformed body reorganization body and releasing propagale stages. The eight developmental stages could be epitomized as three develop mental phases, including the proliferation phase was vigorous cell division and developed into polar or apolar cell musses, the globularization phase was characterized with dermal structure, and the reproduction phase was directly or indirectly releasing propagules. Measuring cell packing volume (CPV) in-dicated that the pro-globular body stage and globularization phase was higher growth rate with 10 days time increase in each subculture generation.
There are two models or three pathways for maintenance and recyclic growth of embryogenic cell culture. The duration of cyclic growth varied in the range 2 ~ 5 months dependent growth phases, refresh medium ratio and sub-culture duration. While the conditional medium：fresh medium of 1:1 ~ 1:4 ratio, and 10 days interval of subculture, would maintain embryogenic cell growth vigorously. The growth type and cyclic pathway were affected by cells exogenous auxin.
The assay of embryogenic potentiality of suspension cells was conducted on SH median supplemented with 0.4 mg/l NAA, 45 g/l sucrose and 2.5 g/l gelrite. If was demonstrated that the cell population of pro-globular body stage and globular body stage showed higher competence of somatic embryogenesis and regenerated an amount of somatic embryos quickly.
The extracellular pH value associated of the three growth phases was measured at the end of subculture generation. The proliferation phase showed pH 5.3±0.3, the globulization phase indicated pH 5.6±0.3 and the reproduction phase was pH 5.35±0.3. The normal cell line''s pH values arose above 5.0 under long-term subculture. The abnormal cell population downgraded to 4.2. It is suggested that the extracellular pH level may serried as indexing of cell growth vigor.
In the subculture generation, the extracellular pH change could be distinguished as rapid change and auto-regulated phases. The rapid rising occurred 1 ~ 2 hours after subculture, then drop to lower pH level by 30 hours. Thereafter, gradually auto-regulated to a higher level until the end of subculture generation, which was associated with growth phase.

內 容 目 次
誌 謝 Ⅰ
縮 寫 字 Ⅱ
內容目次 Ⅲ
圖 目 次 Ⅴ
表 目 次 Ⅸ
一、前 言 1
二、前人研究 3
(一)流蘇的組織培養及超低溫保存： 3
1.胚培養： 3
2.誘導結合子胚之體胚發生： 3
3.細胞懸浮培養及體胚發生： 4
4.細胞超低溫保存： 4
(二)細胞懸浮培養的生長發育： 5
1.懸浮細胞之生長發育及循環生長途徑： 5
2.懸浮細胞之生長分析： 6
3.懸浮細胞之胚性潛力： 6
(三)細胞懸浮培養之pH變化： 7
1.胞內pH值變化： 7
2.胞外pH值變化： 8
三、材料與方法 10
(一)植物材料： 10
(二)流蘇細胞懸浮培養系統之建立： 10
1.誘導胚性癒合組織： 10
2.細胞懸浮培養： 10
3.懸浮細胞增生培養： 10
4.生胚平板培養： 11
(三)流蘇懸浮細胞之生長發育： 11
1.流蘇懸浮細胞生長發育型態之觀察： 11
2.流蘇懸浮細胞循環生長途徑的觀察與分析： 11
3.流蘇懸浮細胞之生長分析： 11
4.流蘇懸浮細胞生長發育之影響因子試驗： 12
5.流蘇懸浮細胞發育期之胚性檢定： 13
(四)流蘇細胞培養pH變化之特性： 13
1.一般胞外pH值的測量與分析： 13
2.胞外pH值電腦監測系統： 13
3.流蘇懸浮細胞培養期間pH值之變化模式： 14
4.流蘇懸浮細胞胞外pH變化之影響因子試驗： 14
四、結 果 16
(一)流蘇細胞懸浮培養之生長發育： 16
1.流蘇懸浮細胞之生長發育期及其生長類型： 16
2.流蘇細胞懸浮培養之循環生長途徑及其週期： 18
3.流蘇懸浮細胞之生長分析： 19
4.流蘇懸浮細胞生長發育之影響因子： 20
5.流蘇胚性懸浮細胞胚性檢定及平板培養生長發育之形態變化： 22
(二)流蘇細胞懸浮培養pH變化之特性： 22
1.流蘇懸浮細胞生長發育期及發育相pH值變化範圍： 22
2.流蘇懸浮細胞繼代培養期間pH之變化模式： 23
3.流蘇懸浮細胞繼代培養期間pH變化之影響因子： 23
3.流蘇懸浮細胞生長自調之pH範圍： 25
五、討 論 26
(一)流蘇細胞懸浮培養之生長發育： 26
1.流蘇懸浮細胞之生長發育及影響因子之探討： 26
2.流蘇細胞懸浮培養之循環生長途徑及其週期： 27
(二)流蘇細胞懸浮培養pH變化之特性： 28
1.流蘇懸浮細胞培養期間pH變化之模式及影響因子之探討： 28
2.流蘇懸浮細胞生長發育期及發育相pH值之範圍： 30
3.流蘇懸浮細胞生長自調之pH範圍： 30
六、中文摘要 70
七、英文摘要 72
參 考 文 獻 74

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