(64(3):177-188)In Vitro Seed Germination and Micropropagation of Rehmannia glutinosa Libosch

本研究利用地黃 (Rehmannia glutinosa Libosch) 種子進行無菌播種，地黃種子以0.6% 次氯酸鈉溶液消毒15 min 可獲得良好之殺菌效果，可明顯降低汙染率，並有最高發芽率，分別為5% 與80%。以無菌播種地黃小苗之葉片與葉柄作為微體繁殖材料，接種於含有3% 蔗糖、0.8% Bacto agar 及0.01 mg L-1 NAA 之MS 培養基 (以下簡稱基本培養基) 並添加0–2.0 mg L-1 BA 不同濃度，分別於光照與黑暗環境下培養。結果顯示，暗處理下培植體有較高之成活率，且葉柄培植體較葉片培植體之再生反應為佳，葉柄培植體於含有2.0 mg L-1 BA培養基中於黑暗環境下培養具有最高之存活率62%，可同時誘導出芽體與癒傷組織，分別為10% 與62%。另以地黃組培苗之頂芽莖段與莖中段2 種不同莖段部位作為微體繁殖材料，接種於添加不同蔗糖濃度 (3–9%)之基本培養基中培養，結果顯示以3% 蔗糖為最佳，成活率可達100%，9% 高蔗糖濃度為最差，會導致培植體褐化死亡。進一步利用組培苗之頂芽莖段、莖中段與近基部莖段3 種不同莖段部位培植體培養於含有3%蔗糖濃度並添加0–2.0 mg L-1 BA 濃度之基本培養基中誘導芽體，3 種培植體中以頂芽莖段表現最佳，成活率為80–100%，BA 濃度於0.5–1.0 mg L-1 之間可誘導出最多芽體，每1 培植體平均可誘導出27 個芽。莖段培植體與BA 濃度二者皆顯著影響地黃芽體之誘導率與培養存活率，且二者間具有交感效應。 In vitro studieswere conducted to establish protocol for micropropagation of Rehmannia glutinosa Libosch, an important Chinese herbal medicine. Results showed that seeds disinfection with 0.6% sodium hypochlorite for 15 min significantly reduced the contamination rate to 5% and obtained the highest germination rate to 80%. Comparing the regeneration rate of leaf and petiole explants from in vitro grown seedlings revealed that petiole explants growing on MS medium containing 0.01 mg L-1 1-Naphthaleneacetic acid with 2.0 mg L-1 Benzyladenine (BA) and cultivated in darkness obtained a better survival rate (62%) along with 10% shoot induction rate and 62% callus induction rate. Cultivating shoot tip and nodal stem segment of in vitro grow seedlings on the basal medium containing various sucrose concentrations (3–9%) showed that addition of 3% sucrose had the highest survival rate (100%). Furthermore, shoot tip, inter-nodal and basal nodal stem segments of seedlings were cultured on the basal medium containing 3% sucrose and various BA concentrations (0.0–1.0 mg L-1) to evaluate their regenerative potential. The results showed that shoot tip had the highest survival rate (80–100%) and had the most adventitious shoots induced per explant when cultured on the medium containing 0.5 mg L-1 BA. The results show that type of stem segment explants and BA concentration are key factors influence the micorpropagation of Rehmannia glutinosa Libosch

Explant Types Derived from Flower Stalk Culture and 6-Benzyladenine Concentrations Affect Shoot Differentiation of Phalaenopsis Hybrid in Subculture

Phalaenopsis (Taisuco Snow × Wataboushi) ‘T343’在可見第一朵花蕾分化時期之花梗節為培植體，進行初代花梗芽誘導，再利用初代花梗節誘導長出之營養芽的短縮莖、葉片、根，以及切除營養芽後保留約2-3 mm 莖基組織之原花梗節，作為繼代培養芽體誘導之材料，接種於含不同濃度(0.0、1.25、2.5、5.0、10.0 及 15.0mg•L-1)苯基腺嘌呤(6benzyladenine, BA)與0.01 mg•L-1 萘乙酸(α-naphthalene aceticacid, NAA)之半量Murashige & Skoog 基本鹽類培養基中8 週。試驗結果顯示，保留少量莖基組織之原花梗節培植體，其繼代培植體分化率在71%-100%之間，隨著BA濃度的提升，平均最高可誘導4.8 個芽體；BA 濃度除對增殖芽數有顯著影響外，對芽體分化之型態亦有顯著影響，誘導產生之芽體型態可分為單一營芽、叢生營養芽、芽體與擬原球體(protocorm-like bodies, PLBs)共存，以及芽體與綠色團塊類癒合組織共存等四種。以營養芽去除葉片及根部後之短縮莖作為繼代培養之培植體，於0.0-1.25 mg•L-1 BA 中，有較高的根誘導率，為50%-70%，隨著BA 濃度提升至5.0-10.0mg•L-1，分化型態以芽體為主，平均誘導之芽體數可達3.3-3.5 個。以營養芽上長出之葉與根作為培植體，在0.0-10.0 mg•L-1 BA 的濃度下，葉片可誘導出PLBs，根可誘導出癒傷組織(callus)之分化型態，誘導率於10.0 mg•L-1 BA 之培養基中達最高，其PLBs 與callus 誘導率分別為20%與8%。 Flower-stalk nodes of Phalaenopsis (Taisuco Snow × Wataboushi) ‘T343’, taken at the developmental stage when the first flower bud was visible, were used as explants in tissue culture. Four types of explants were subsequently taken from the induced buds of the initial flower stalk culture. They were leaves, roots,leafless-basal-stems of the new buds, and the original flower-stalk nodes containing 2-3 mm basal stems after removing the new buds. Explants were then cultured on a 1/2MS basal salt medium containing 0.01 mg•L-1 NAA and various concentrations of BA (0.0, 1.25, 2.5, 5.0, 10.0 and 15.0 mg•L-1) for 8 weeks. Those explants of original flower nodes with 2-3 mm basal stems had 70% to 100% survival rates after the subculture. The number of shoots produced from each explant increased with BA concentration increase, and 4.8 shoots per explant was the highest. Besides the effect on shoot number, BA concentration also had a significant influence on the type of shoot differentiation. There were four differentiation types, namely: single vegetative shoot, multiple vegetative shoots, shoot plus PLBs, and shoot plus callus-like tissue. For the explants of leafless-basal-stems of the new buds, the highest root induction rate was 50% to 70%, which was at 0.0 or 1.25 mg•L-1 BA level. At higher concentrations of BA, more adventitious shoots were produced. They had 3.5 shoots per explant at 10.0 mg•L-1 BA. When leaves and roots were used as explants, PLBs and callus were developed respectively. The former had 20% and the latter had 8% of induction rate at 10.0 mg•L-1 BA

(65(4):384-394) Influence of Explant, Plant Growth Regulator and Illumination on Adventitious Shoot Induction of In Vitro Cultured Salvia miltiorrhiza

本研究利用丹參 (Salvia miltiorrhiza) 組織培養苗之葉柄和葉片培植體進行不定芽的誘導試驗。葉柄培植體於含有1 mg L-1 芐腺嘌呤 (N6-benzyladenine; BA) 與0.5 mg L-1 奈乙酸 (α-naphthaleneacetic acid; NAA) 之MS (Murashige & Skoog 1962) 培養基中培養6 wk 後不定芽形成率達100%，平均每個培植體可形成3.7 個不定芽。將葉柄和葉片培植體預培養於含有1–2 mg L-1 2,4-二氯苯氧乙酸 (2,4-dichlorophenoxyacetic acid; 2,4-D) 之MS培養基2 或3 wk 後，再分別繼代培養於不含植物生長調節劑之MS 培養基中培養4 或3 wk，可誘導癒合組織和不定根形成。葉片癒合組織於含有0.25 mg L-1 BA 與0.2 mg L-1 2,4-D 之MS 培養基於黑暗中培養8 wk，顯示除了癒合組織增殖外，亦可形成少數不定芽。將癒合組織繼代培養於含有相同2,4-D 濃度但不同BA 濃度之培養基中，於光照或黑暗環境培養8 wk 後，其中以2 mg L-1 BA 配合光照處理可產生最多不定芽，每接種0.2 g 癒合組織平均可形成14.1 個不定芽。本研究利用丹參組織培養苗建立直接與間接不定芽再生大量繁殖體系，除可供生產丹參種苗所需外，亦可應用於誘變與轉基因之研究。 Petiole and leaf explants derived from in vitro Salvia miltiorrhiza were used for shoot regeneration in this study. The highest adventitious shoot formation rate of 100% with 3.7 shoots/explant in average was obtained from petiole segments cultured on Murashige and Skoog’s (MS) medium containing 1 mg L-1 N6-benzyladenine (BA) and 0.5 mg L-1 α-naphthaleneacetic acid (NAA) for 6 wk of culture. Callus and adventitious roots were induced from petiole and leaf segments pre-cultured on the MS medium supplemented with 1–2 mg L-1 2,4-dichlorophenoxyacetic acid (2,4-D) for 2–3 wk followed by transferring on a hormone-free MS basal medium for a total 6 wk of culture. Calli were proliferated on the MS basal medium containing 0.25 mg L-1 BA and 0.2 mg L-1 2,4-D under darkness for 8 wk of culture along with few adventitious shoots were found. Proliferated calli were subcultured to the medium containing with same concentration of 2,4-D in combination with various BA concentration under light and dark condition for shoot regeneration. The highest induction number of adventitious shoots was 14.1 shoots 0.2 g-1 callus from the medium containing 2 mg L-1 BA under light condition. An efficient micropropagation system of Salvia miltiorrhiza by direct and indirect adventitious shoot regeneration systems were established in this study which would not only supply for plantlet production but also apply on mutation and genetic transformation studies

Influences of Cytokinins and Antibiotics on Shoot Regeneration of Chrysanthemum Cultivars

本研究以多種菊花栽培品種首先建立其無菌瓶苗培養，再利用瓶苗之葉片及葉柄為再生試驗培植體，以不同生長調節劑組合處理添加於培養基，測試各品種的再生反應。結果顯示品種間芽體再生能力差異顯著，同一品種之葉片或葉柄培植體再生率則無不同。之後應用再生反應較佳之大花栽培種‘ 黃秀芳’進行抗生素試驗，三種抗生素 carbenicillin、cefotaxime 及 timentin 的添加對葉柄培植體芽體之再生皆未產生明顯的抑制作用，其中 200 mg/L timentin 處理再生率最高。 The study tested the regeneration of various chrysanthemum cultivars by establishing its growth cultured in vitro, then use in vitro cultured petiole or leaf as regeneration explants. The different mix of plant growth regulator treatments were added at medium, and investigated for shoot regeneration. The results showed that regeneration ability varied among tested cultivars, however no difference shown on regeneration rate between leaf and petiole types of explants in the same cultivar. Later apply three antibiotics carbenicillin, cefotaxime and timentin with various concentrations to precede antibiotic test with the “HuangXiuFang” petiole explants of better regeneration. All three antibiotics had no obvious suppressing influence on shoot regeneration, and among them, the treatment of 200 mg/L timentin had highest regeneration rate