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Influence of ventilation closures on plant growth parameters, acclimation and anatomy of leaf surface in Scrophularia yoshimurae Yamazaki - a medicinal plant native to Taiwan
No full textDifferent ventilation closures, including aluminum foil (AF) and a varying number of dispense papers (DP) had different effects on leaf growth parameters, in vitro rooting, survival rate and the anatomical features of the leaf surface of in vitro and ex vitro acclimated plants of Scrophularia yoshimurae—an important medicinal plant. The lowest plant growth parameters and ex vitro acclimation rate (<7.0%) were obtained using AF as ventilation closure. A scanning electron microscopy (SEM) study of leaf surfaces of plants derived from different ventilation closure treatments showed that parameters—including density and size of epidermal cell and stomata, size of guard cells, and stomata aperture-differed significantly among various treatments, and this in turn affected plant survival rate. Leaves derived from AF treatment had higher epidermal cell (15094 cells/mm2) and stomata (38/mm2) densities than DP treatments. Well-ventilated container closures, such as with DP, improved the morphological characteristics of leaves and in turn enhanced the survival rate during ex vitro acclimation (maximum rate being 66.7%). The present study not only provides an improved micropropagation method of S. yoshimurae but also gives scientific reasons for the different acclimation rates obtained with various container closures
(65(4):384-394) Influence of Explant, Plant Growth Regulator and Illumination on Adventitious Shoot Induction of In Vitro Cultured Salvia miltiorrhiza
No full text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
(56(1):21-30)In Vitro Plantlets Acclimation and Tanshinones Analysis of Salvia Miltiorrhiza
No full text本研究探討吲哆丁酸 (indole-3-butyric acid; IBA) 對丹參 (Salvia miltiorrhiza) 組培芽體 (in vitro shoot) 發根之影響，以及培養容器封口透氣資材與馴化條件對組培苗移植存活率關係，並分析成株丹參酮之含量。組培芽體於添加0.5 mg L-1 IBA之半量Murashige and Skoog基本鹽類 (1/2MS) 培養基，利用鋁箔紙封口培養2週後，再以三層藥包紙進行封口置換處理 (AF2+DP4) ，其發根率可達90%。將組培苗 (plantlet) 移植於已滅菌之BioMix培養土：蛭石：珍珠石 = 1：1：1 (v/v) 混合介質，再以透明塑膠袋覆蓋植株培養於日夜溫為22℃，10h / 18℃，14h、光照強度約60 μmol m-2 s-1之生長箱進行馴化處理，透氣預處理組培苗存活率可達80.1%，較持續以鋁箔紙封口培養前處理組培苗存活率則只有27.8%。此外，經由高效率液相層析儀 (high performance liquid chromatography；HPLC) 分析丹參各檢品之丹參酮類 (tanshinones): tanshinone-I (Tan-I), tanshinone-IIA (Tan-IIA), cryptotanshinone (Crypto) 含量結果顯示，組培苗經田間栽植9個月後植株根部（紅根）之Tan-I＋Tan-IIA＋Crypto總含量約1.85 mg g-1 dw，為市售丹參藥材的1.5倍。An efficient micropropagation protocol for producing high quality plantlets that are easy to macclimatize in field was developed for Salvia miltiorrhiza. In vitro grown shoots induced rooting (90%) after culturing on half-strength Murashige and Skoog’s basal medium (1/2MS) supplemented with 0.5 mg L^(-1) indole-3-butyric acid (IBA). Culture vessels were capped with aluminum foil for 2 weeks, and thereafter replaced with 3 layers of dispense paper for 4 weeks (AF2+DP4) were better than controls capped with aluminum foil for 6 weeks (AF6). Rooted shoots were transplanted into plastic pot containing a mixture of BioMix: vermiculite: perlite (1: 1: 1 ratio) (v/v). For acclimation, these pots were covered with transparent plastic bags and kept in a growth chamber under 14 h light at 22℃ and 10 h dark at 18℃ for 4 weeks. At 4 weeks of observation, these conditions resulted in the highest survival rate of 80.1% compared to AF6 treatment (27.8%). To compare, tanshinones contents (tanshinone-I, tanshinone-IIA and cryptotanshinone), the bioactive compounds in Salvia miltiorrhiza were estimated in the aerial parts and roots between in vitro micropropagated plants and market crude drug of S. miltiorrhiza by high performance liquid chromatography (HPLC). The quantity of tanshinones in red roots of in vitro micropropagated plants was measured as 1.85 mg g^(-1) dw about 1.5-fold higher than market crude drug
Explant Types Derived from Flower Stalk Culture and 6-Benzyladenine Concentrations Affect Shoot Differentiation of Phalaenopsis Hybrid in Subculture
No full textPhalaenopsis (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
(64(3):177-188)In Vitro Seed Germination and Micropropagation of Rehmannia glutinosa Libosch
No full text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
(53(4):249-260)Influence of Salt Strength, Sucrose, Auxins and Container Closure on Root Formation and Acclimation of In Vitro Bupleurum kaoi Plantlets
No full text本研究探討培養基組成中之MS (Murashige & Skoog)鹽類濃度、蔗糖濃度與植物生
長素，對高氏柴胡(Bupleurum kaoi Liu, Chao et Chuang)組培苗發根之影響，並藉由瓶口
覆蓋物處理降低瓶內溼度，以提高瓶苗移植存活率之方法，建立高氏柴胡組織培養之大
量繁殖種苗體系。結果顯示，低MS 鹽類濃度(1/4MS 與1/2MS)及3%蔗糖培養基之組
培苗根系形成較佳且存活率較高。組培苗培養於含有0.5 mg/l 丁酸(indole-3-butyric
acid; IBA)之1/2MS 培養基，發根率可達100%，且組培苗不會產生基部癒合組織化現
象。利用鋁箔紙覆蓋培養2-3 週後，再以三層藥包紙進行覆蓋物置換處理之瓶苗與根系
均生長良好，瓶苗移植存活率約為76%，較高於鋁箔紙覆蓋培養6 週之處理(約33%)。
將組培苗培養於含0.5 mg/l IBA 與0.1-0.2 mg/l 奈乙酸(α-naphthaleneacetic acid; NAA)
之1/2MS 培養基，於培養2 週後再進行藥包紙置換處理，可形成具有較粗根徑之組培
苗，將出瓶組培苗除去部分葉片後剩餘長約3-4 cm 之瓶苗，移植於已滅菌之培養土
(BioMix)：蛭石：珍珠石 = 1：1：1 (v/v)混合介質，育苗盤放入有蓋透明塑膠盒後置於
日夜溫為22℃(10 h) / 18℃(14 h)、光照(約60 μmol/m2/s)之生長箱進行馴化處理，4 週
後之存活率高達92%，顯示添加少量NAA 有利於高氏柴胡組培苗之發根與移植存活率
的提昇。本研究利用組培苗瓶內處理(改善培養基組成與培養容器瓶口覆蓋物)及出瓶後
馴化處理(有蓋透明塑膠盒)之結果，將有助於高氏柴胡組培苗產業化生產系統之建立。An efficient protocol for producing high quality rooted plantlets which are easy to acclimatize for field
growth was established for micropropagation of Bupleurum kaoi Liu, Chao et Chuang. In this study, the
medium containing half- and quarter- strength of MS basal salts (1/2MS and 1/4MS) and 3% (w/v) sucrose
was found suitable for shoot survival and root formation of in vitro B. kaoi plantlets. A 100% root
formation rate was obtained from in vitro shoots cultured on the medium containing half-strength MS salt,
0.5 mg/l indole-3-butyric acid (IBA) without basal callus further induced. An increased survival rate for
in vitro plantlets acclimation by using ventilative container closure of dispense paper to exchange the
normal used aluminum foil after 2-3 weeks culturing in a total 6 weeks incubation span was achieved.
Leaves of in vitro rooted plantlets were partial trimmed (3-4 cm remnant in length) and transplanted into
BioMix: vermiculite: perlite = 1: 1: 1 (v/v) mixed substrate inside a transparent plastic box with cover.
Boxes were put into a growth chamber under 22℃ (14 h in light) / 18℃ (10 h in darkness) environmental
condition for 4 weeks acclimation. The best survival rate was 92% after 4 weeks of acclimation from
plantlets previously cultured on half-strength MS medium containing with 0.5 mg/l IBA and 0.1-0.2 mg/lα
-naphthaleneacetic acid (NAA), in combination with changing aluminum foil with 3 layers of dispense
paper as container closure after 2 weeks culturing. These results including in vitro treatment by modifying
medium components and exchanging container closure for root formation and plantlets quality; ex vitro
treatment by planting plantlets inside a transparent plastic box with cover for reducing transpiration would
be useful for industrial production of in vitro Bupleurum kaoi plantlets
(67(1):44-53)Effects of Two-Stage Culture and Paclobutrazol on In Vitro Bulblet Growth and Rooting of Hippeastrum hybridum
No full text本研究利用孤挺花 (Hippeastrum hybridum Hort.)「台農1 號-紅粉佳人」(‘Tainung No.1-Pink Lady’)、「紅獅」(‘Red Lion’) 及「千禧之星」(‘Blossom Peacock’) 組培苗為材料，探討兩階段培養及巴克素對鱗莖生長與發根之影響。「台農1 號」鱗莖 (直徑約5 mm) 於含有0.1 mg L-1 苯甲基腺嘌呤 (benzylaminopurine; BA) 與0.1 mg L-1 奈乙酸 (α-naphthalene acetic acid; NAA) 之液態或固態MS培養基中培養8 wk (第一階段)，再接種至相同組成之固態培養基8 wk 後 (第二階段)。結果顯示，液-固態兩階段培養具有顯著增加組培苗鮮重、葉數、葉長及根數之效果。利用「紅獅」與「千禧之星」鱗莖 (直徑約10 mm) 進行巴克素 (Paclobutrazol; PBZ) 液態培養試驗，結果顯示，對照組鱗莖培養至8 wk 時，已有部分組培苗出現嚴重褐化的現象，建議繼代培養間隔時間不宜超過8 wk。「紅獅」組培鱗莖在50–75 mg L-1 PBZ 培養條件下，PBZ 處理之根數顯著較多於對照組，但PBZ 處理間並無顯著差異。然而，「千禧之星」鱗莖在25–75 mg L-1 PBZ 處理時根系生長受到抑制，且濃度越高時根數就越少，而75 mg L-1 PBZ 處理則較對照組具有顯著較短與較寬的葉片；將PBZ 濃度降低至5 mg L-1，則有增大鱗莖與促進發根之效果。
An improved method for in vitro bulblet growth and rooting of Hippeastrum hybridum was established in this study. In vitro bulblets of Hippeastrum hybridum ‘Tainung No. 1’, ‘Red Lion’, and ‘Blossom Peacock’ were used as materials to conduct the liquid-solid or solid-solid two-stage culture, and additional paclobutrazol was added into culture medium to enhance growth and rooting of in vitro Amaryllis bulblets. In vitro bulbs (5 mm diameter) of ‘Tainung No. 1’ were cultured in liquid or solid MS medium containing 1 mg L-1 BA and 0.1 mg L-1 NAA for 8 wk of culture before transferring to a solid MS medium with the same components for another 8 wk of culture. The results showed that liquid-solid culture was superior to solid-solid culture on fresh weight of bulblets, leaf number, leaf length, and root number. In vitro bulblets of ‘Red Lion’ and ‘Blossom Peacock’ showed severe browning or death after 8 wk of culture on the control medium. Therefore, it is suggested that the subculture interval should not exceed 8 wks. The root number of ‘Red Lion’ bulblets by 50–75 mg L-1 PBZ treatment was found significantly higher than that of control. No significant difference among PBZ treatments was found. However, root numbers of ‘Blossom Peacock’ were reduced treated with 25–75 mg L-1 PBZ concentrations. Leaf with shorter and wider shape was observed in 75 mg L-1 PBZ treatment. However, a lower concentration of 5 mg L-1 PBZ was found beneficially to bulb diameter, root number and root length
(68(2): 137-147)Effect of Explant Size, Sucrose and Activated Charcoal on In Vitro Plantlet Proliferation and Growth in Amaryllis
No full text本研究以孤挺花「台農1 號」(Hippeastrum hybridum Hort. ‘Tainung No. 1’) 組培苗為材料，探討培植體大小、蔗糖及活性炭濃度對組培苗生長之影響。以鱗莖直徑約3、6、9 mm 之4 分縱切鱗莖作為培植體進行培養，結果顯示不同大小培植體之組培苗增殖率並無顯著差異。每個1/4 鱗莖於培養8 wk 後，約可形成1.1−1.3株組培苗。此外，9 mm 處理之培植體，在8 wk 培養後可獲得具有較大鱗莖之組培苗；然而，在延長培養時間至12 wk 後，9 mm 與6 mm 處理間已無顯著差異。利用鱗莖直徑約3−5 mm 之組培苗作為培植體，進行1.5−9.0% 蔗糖與0−2 g L-1 活性炭濃度組合之複因子試驗，結果顯示6.0% 蔗糖配合1.0−2.0 g L-1 活性炭處理，對於增加組培苗鱗莖直徑與鮮重較為有利，分別達到8 mm 與1.73−1.75 g；但是當蔗糖濃度達到9% 時，則會抑制組培苗之生長。就組培苗根數而言，在含有6% 蔗糖搭配0.5−2.0 g L-1 活性炭條件下，可形成8.8−9.6條根，均較高於其他處理。就組培苗葉數而言，於0.5−2.0 g L-1 活性炭濃度下，蔗糖濃度在1.5−6.0% 之間並無顯著差異。綜合而言，蔗糖濃度自1.5% 提高至6.0%，可增加組培苗之鱗莖直徑、鮮重及根數，若同時添加適量活性炭，則具有協同增效蔗糖之效果。
Factors of explant size, concentration of sucrose and activated charcoal in the culture medium were investigated on in vitro growth of Hippeastrum hybridum Hort. ‘Tainung No. 1’ plantlet in this study. One-quarter vertical cut bulb explants derived from 3, 6 and 9 mm diameters of bulb were evaluated after 8 wk and 12 wk of culture. Proliferation rate of 1.1−1.3 plantlets per one-quarter bulb explant was obtained after 8 wk of culture, and no significant difference was found among all treatments. Furthermore, plantlets with larger bulb were obtained from explants derived from explants of 9 mm treatment. However, no significant difference on bulb diameter of 6 mm and 9 mm treatments was found after extending culture time to 12 wk of culture. A factorial experiment using 1.5−9.0% sucrose in combination with 0−2.0 g L-1 activated charcoal was conducted using explants derived from plantlets with 3−5 mm diameter of bulb. The better results were obtained from 6% sucrose in combination with 1.0−2.0 g L-1 activated charcoal on increase of bulb diameter and fresh weight which were 8.0 mm bulb diameter and 1.73−1.75 g fresh weight per plantlet, respectively. It was also found that growth of plantlets was inhibited in the medium containing 9.0% sucrose. In terms of the root number of plantlet, the best results of 8.8−9.6 roots per plantlet were obtained from 6.0% sucrose in combination
with 0.5−2.0 g L-1 activated charcoal, and higher than the other treatments. For the leaf number of plantlet, no significant difference was found by using 1.5−6.0% sucrose in combination with 0.5−2.0 g L-1 activated charcoal. In summary, raising sucrose concentration from 1.5% to 6.0% would increase bulb diameter, fresh weight and root number of plantlet, and adding proper concentration of activated charcoal would has a synergistic effect with sucrose
(66(3):184-192)Development of Microspore Embryogenesis and Plant Regeneration of Brassica oleracea var. italica
No full text十字花科之青花菜 (Brassica oleracea var. italica) 是營養價值很高的蔬菜，本研究以台灣青花菜商業一代雜交品種 (F1) 為材料，測試不同F1 之小孢子培養反應，並建立小孢子之胚誘導及胚發芽所需條件，用以生產雙單倍體 (doubled haploid; DH) 植株作為培育純系之用。利用5 種F1 青花菜以每毫升含有4 × 104 個小孢子濃度接種於NLN-13 培養液中，並以32℃處理1 d，其中3 品種有胚形成，2 品種未有反應，胚誘導率以「慶農B-45」最高。比較取自「農友天綠」不同長度花蕾之小孢子，結果顯示取自4.0–5.0 mm 長度花蕾之小孢子，平均可獲得17.1 個胚/皿，顯著較取自3.0–3.9 mm 長度花蕾之7.0 胚/皿為高 (P < 0.01)。「慶農B-45」以含有13% 蔗糖之NLN 培養基 (全量及半量鹽類濃度) 與32℃處理時間 (1、2 及3 d) 組合進行小孢子培養。結果顯示以全量NLN 培養基在32℃處理1 d 獲得6.9 胚/皿最高。將「慶農B-75」子葉期胚施以1–5 d 之脫水配合溫度處理，處理後之胚先接種於含有生長調節劑之固態培養基預培養1 wk，再繼代於不含生長調節劑之培養基中培養4 wk 後，調查胚發芽之比率。結果顯示子葉期胚於25℃以濾紙脫水1 d 或於4℃以濾紙脫水1–5 d 皆有助提高發芽率 (35.0–48.3%)。將「慶農B-75」由小孢子發育而來之23 株小苗以流式細胞儀檢測其染色體倍體性，結果顯示DH 及單倍體之比率分別為60.9% 及34.8%。DH 植株之比率超過60%，有助於育種實務上之利用。
Brassica oleracea var. italica is an important vegetable in the world with rich nutrition for human health. The objective of this study was to establish a microspore culture system using elite broccoli F1 hybrids from Taiwan to produce doubled-haploid (DH) lines for breeding purpose. Microspores taken from 5 commercial F1 hybrids were surveyed for embryogenesis. Microspores were inoculated with 4 × 104 microspores/mL into NLN-13 medium in combination with a heat shock treatment at 32℃ for 1 d before moving to 25℃ for culturing. Embryo induction was found derived from 3 out of 5 cultivars, among them ‘Chinglong B-45’ had the highest embryogenesis rate. However, two cultivars were found without any embryogenesis. Microspores of ‘Known-you Tender green’ were taken from two different bud lengths for embryogenesis. A higher embryo induction rate (17.1 embryos/ petri dish) was obtained from 4.0–5.0 mm length of bud than that of from 3.0–3.9 mm length of bud (7.0 embryos/petri dish). An experiment using two culture media in combination with three durations at 32℃ was conducted using ‘Chinglong B-45’. Among six treatments, NLN medium with 1-d culturing at 32℃ had the highest embryogenesis with 6.9 embryos/petri dish. Various stress treatments were conducted using cotyledonary embryos derived from ‘Chinglong B-75’ to improve germination. Treated embryos were precultured on the hormone containing medium for 1-week followed by 4 wk culturing on a hormone-free medium for shoot germination. A low shoot germination rate of 1.7% was obtained from the control which was without any stress treatment. In contrast, shoot germination rate of 46.7% and 48.3% were obtained from filter paper dry treatments at either 25℃ for 1 d or at 4℃ for 5 d, respectively. Ploidy levels were analyzed using flow cytometry for a total of 23 microspores derived plants of ‘Chinglong B-75’. Rates of 34.8% and 60.9% were obtained for haploid and diploid, respectively. A high DH production rate of 60% derived from the microspore culture would facilitate breeding program in practice
(67(1):94-105)Screening on Tanshinone Production and Time Course Culture of Tetraploidy Hairy Roots of Salvia miltiorrhiza
No full text丹參 (Salvia miltiorrhiza) 為唇形科多年生草本植物，是中醫治療心血管疾病的重要藥材。4 倍體 (4N) 毛狀根與其2 倍體 (2N) 相較，具有二次代謝物含量較高之特性，是離體培養大量生產植物二次代謝物的良好工具。本研究利用秋水仙素誘導所得之4N 丹參毛狀根群，探討從中篩選出高丹參酮產量毛狀根系 (以下簡稱根系) 的可行性。首先以固體培養基進行連續3 次、每次8 wk 的根系培養，並依據丹參酮產量從103 個4N 根系中篩選出前32 名，繼續進行連續3 次、每次8 wk 的液體培養；同時從32 個4N 根系中挑選出2 個根系與2N 根系一起進行時序培養。固體培養的結果顯示，4N 根系群的平均生質量較2N 根系低，僅達2N 根系的67.2%，但4N 根系群的平均總丹參酮含量較2N 根系為高，是2N 根系的4.66 倍，且4N 根系群的平均丹參酮產量為2N 根系的3.29 倍。檢視4N 根系群丹參酮含量比例的變化，以隱丹參酮含量之比例增加最多，丹參酮I 含量之比例減少最多，丹參酮IIA 含量之比例減少其次。在固體培養中丹參酮產量表現最佳之前10 名4N 根系，在液體培養篩選後僅餘2 根系仍保持於前10 名，顯示4N 根系在固、液體培養中的表現並非完全一致，建議在固體培養篩選階段保留較多的根系，作為後續液體培養篩選之用。時序培養結果顯示，4N 根系的生長速度較2N 根系慢，2 個4N 根系的生長模式亦存有差異；2 個4N 根系之丹參酮含量皆於第8 週達到最高，較2N 根系於第12 週達到最高明顯提早。綜合來看，雖然4N 根系之生質量平均較2N 根系為低，但因4N 根系具有丹參酮含量較高，且丹參酮產量累積較快之特性，因此在丹參酮的生產效率上，4N 根系仍較2N 根系為高，且透過篩選機制可將高產之4N 根系選拔出來，作為大規模培養之基礎。
Salvia miltiorrhiza Bunge, a perennial herb of Labiatae Family, has been used to cure cardiology disease in traditional Chinese medicine for long history. Polyploidy hairy roots containing higher concentration of secondary metabolites than its diploidy are considered as better explants for in vitro production of valuable secondary metabolites. The objective of this study was to establish a selection strategy on tanshinone production of tetraploidy hairy root lines (4N lines) which were induced from the diploid hairy root line (2N line) by colchicine treatment. Three consecutive cultures in solid medium were first conducted on 103 lines of 4N. Among them, 32 lines with the highest tanshinone production were selected for another 3 consecutive liquid medium culture. Meanwhile, two 4N lines and the 2N line were used to conduct the time course culture in liquid medium for 12 wk span. Screening results from solid cultures showed that the average biomass of 103 lines of 4N was only counted 67.2% of the 2N line. However, average total tanshinone contents and productivity of 4N lines were counted 4.66 times and 3.29 times higher than that of the 2N line, respectively. Composition ratios of cryptotanshinone, tanshinone I, and tanshinone IIA contents on 4N lines and 2N line were compared. It was found ratio of cryptotanshinone increasing the most in contrast decreasing ratios of tanshinone I and tanshinone IIA. The ten best lines of 4N selected from solid medium on production of tanshinones were found having only 2 lines remaining in the best ten after liquid medium selection. In order not to miss elite lines, lower selection strength was recommended in the stage of solid culture on 4N lines. Results of time course culture showed that lagging growth of 4N lines than that of 2N line. Moreover, two 4N lines had slightly different growth rates. The highest tanshinone contents were found after 8 wk of culture on 4N lines which was much earlier than that of 12 wk of culture on 2N line. In conclusion, although 4N lines have lower biomass than that of the 2N line, 4N lines have higher tanshinone contents and shorter culture period on tanshinone accumulation which would provide better efficacy on tanshinone production than that of 2N lines. It is thought that selection of elite 4N lines will provide a good foundation for in vitro production secondary metabolites
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