黛粉葉腋芽培養與體胚發生再生系統之建立

Establishment of regeneration system via axillary bud culture and somatic embryogenesis in Dieffenbachia spp.

沈榮壽(Rong-Show Shen)

指導教授 ： 李哖

國立臺灣大學/生物資源暨農學院/園藝學研究所/博士(2006年)

https://doi.org/10.6342/NTU.2006.02800

摘要

為增加黛粉葉微體繁殖培植體供給來源與解決內生污染，選用兩個優良品系之成熟莖段，預處理BA進行潛伏芽體誘發，顯示潛伏芽體之誘發隨著BA濃度(0-750 mg/L)增加而遞增；誘發側芽增加數，複莖性品系(Dieffenbachia Chiada-84 ＃1012)比單莖性品系(Dieffenbachia Chiada-84 ＃0804)有較多芽體數，處理濃度以750 mg/L BA 效果最佳。而抗生素預處理對黛粉葉腋芽培植體離體培養污染之影響，未經抗生素處理者幾近全數污染。然以100 mg/L Rifampicin + 100 mg/L Gentamycin 或 100 mg/L Rifampicin + 100 mg/L Gentamycin + 150 mg/L Streptomycin組合濃度處理者，具有顯著降低污染率的效果分別為60%及50%；克服內生菌污染而建立初代無菌培養之腋芽培植體，芽體再生率僅達70%-75%，惟平均每一腋芽培植體芽體增殖數不多為1.2-1.4個，可知經抗生素處裡形態發生潛能降低，再生芽體之生長也顯現抑制現象。
而在黛粉葉無病原培植體準備、無菌培養建立與芽體增殖系統之建構顯示，莖頂分生組織培養於MS基礎培養基含1 mg/L NAA及1 mg/L TDZ培養15週後，最小之莖頂（0.1 x 0.25mm）培植體具有20％的存活率，較大之莖頂生長點（0.90 × 1.20mm）培植體，則顯現高繁殖潛力獲得12.3個再生芽體；另以4種大小黛粉葉腋芽分生組織為培植體來源，剝除3或4片芽鞘數的存活率可達30-35％，同時也顯現剝掉1片芽鞘的生長點培植體平均每一個反應培植體可達11.2個再生芽體；此等結果顯現利用腋芽生長點作為無病原培植體來源可降低黛粉葉內部感染，建立健康分生苗再生體系。此外，以人工乾旱逆境6週枝梢腋芽，進行精細解剖切取腋芽分生組織培養。顯示剝除2至4片芽鞘之生長點培植體，其存活成功率可達80-92.5％，有效降低內生病原菌感染之應用價值。另於培養方式與芽體增殖試驗可知，次級母莖培植體在TDZ濃度為1、5或10mg/L之液體培養者，顯著具有較高的芽體產量，平均芽體形成數可達5-6倍以上的繁殖潛力；而2節短莖培植於液體培養者，其芽體形成繁殖率僅達2倍左右，但芽體生長呈現較為整齊一致；提供黛粉葉微體繁殖種苗量化增殖方式之重要參考。
此外，利用短暫浸漬系統建立快速有效率的黛粉葉微枝梢大量生產體系。顯示以微體繁殖再生枝梢之2節短莖或母莖作為培植體，在MS培養基含1mg/L NAA與0.1-5.0mg/L TDZ或0.1-5.0 mg/L BA組合，在短暫浸漬系統培養容器中裝入330mL液體培養基可誘導產生大量叢生枝梢。尤其在培養基含有5mg/L TDZ之處理組，平均一個2節短莖或母莖培植體分別可得24.5及59.2個枝梢再生，此一產量為傳統固體培養微枝梢產量的3.3-8倍。利用短暫浸漬系統在微枝梢的生產上產生快速且大量的經濟效益，有助於黛粉葉微體繁殖微枝梢量產體系的建立。叢生枝梢經馴化出瓶，以500 mg/kg NAA粉劑預處理，扦插於穴盤噴霧加濕環境，枝梢長度大於3.0㎝之微插穗扦插後30天存活率達80-100％；而叢生枝梢團塊分割成1/4分割或1/2分割處理者，亦得83.3-100％存活率，並可於30天發根期長成緊密充實之株型，可利用於黛粉葉優質採穗母株建立。
另外為了了解生長調節在黛粉葉雄花序培植體體胚發生與植株再生的效果。雄花序培植體培養於修改之1/2 MS含2％蔗糖、1％葡萄糖與0.18％水晶洋菜的固體培養基，顯示在0.5 mg/L Kinetin組合2,4-D為4.0 mg/L或5.1 mg/L等觀察到較高胚狀體發生率為26.7％；根據胚狀體形成率分析黛粉葉體胚發生潛能，顯示2,4-D濃度的增加，與黛粉葉體胚發生頻率呈現顯著相關（R2=0.94）。再依前項結果修改生長素為2,4-D、 Dicamba及Picloram等生長素參試，可知Dicamba、Picloram在濃度為2mg/L培養基，培養第8週能夠獲得強胚性癒傷組織發生，其中2mg/L Dicamba達到指數 IV的胚性癒傷組織指數為72.1％為最高，在12週培養可見30％的成熟體胚形成，平均每個雄花序培植體可得22.2體胚，顯著高於其它處理別。再經修改生長調節劑以2,4-D 4mg/L組合TDZ 0.5-1.0mg/L之處理，進一步誘致高頻率球狀體胚發生率達100％，平均產生32.1-38.2個成熟體胚，少數成熟體胚偶發性轉化獲得再生小植株。
因此，為了更有效提升黛粉葉體胚發生量化增殖與胚苗轉化。雄花序培植體於半量MS基本鹽類，添加20 g/L蔗糖、10 g/L葡萄糖及1.8 g/L Gelrite等為基礎培養基，顯示在2 mg/L Dicamba及0.5 mg/L TDZ組合，具有最高初級體胚發生頻率(100％)，以及最多盾狀體胚形成數達75.2個，對黛粉葉雄花序培植體高效率體胚發生的促進，具顯著的效果。再以前一試驗均質初級體胚，作為次級培植體來源，繼代培養於相同基本培養基添加2 mg/L Dicamba及TDZ（0.5、1mg/L），培養13週後顯現最高效率(100％)的次級體胚發生頻率，次級球狀體胚形成數則達34.3及33.0個，顯示此等培養基組成，有效的促進次級體胚發生和體胚量化增殖。此外，在1/2 MS 添加150 mL/L椰子水之培養基組成，成熟次級體胚(子葉鞘胚)之胚苗轉化試驗顯示，大小約為4-5 mm之鞘葉胚，培養在0.5%葡萄糖及1.0 mg/L TDZ濃度之胚苗轉化率最高達73.3％；平均每一培植體胚苗轉化獲得之再生植株可達7.8個，且可發育成為具形態雙極性之獨立個體。胚苗轉化再生植株，經過馴化出瓶種植之穴盤苗可在溫室中表現正常生育。

關鍵字

黛粉葉 ； 微體繁殖 ； 腋芽形成 ； 體胚發生

並列摘要

In this research the influence of pathogen-free explant preparation on aseptic culture and shoot proliferation in Dieffenbachia ‘Starshine’ was study. Using different sizes of apical meristem as initial pathogen-free explants and cultured on solidified Murashige and Skoog (MS) medium with 1mg/L NAA and 1mg/L TDZ for 15 weeks. The 20% of survival rate were obtained from the minimum size (0.1x0.25mm) of shoot tip explant, and 12.3 shoots were regenerated from the larger size (0.9x1.2mm) of shoot tip explant during primary aseptic culture. Moreover, tests used 4 sizes of axillary bud meristem as initial pathogen-free explant for primary aseptic culture. Result demonstrates that removed 3 or 4 coleoptiles of axillary bud explant, gave the survival rate of 30-35%. But, on average, 11.2 shoots regenerated from excided 1 coleoptile of axillary bud explant. These results indicated using apical meristem as pathogen-free explant gave optimize information for primary aseptic culture in Dieffenbachia ‘Starshine. Furthermore, pretreatment of stock plants with 6-week drought stress and further dissected axillary bud under aseptic condition as pathogen-free explants for primary aseptic culture. Results show the explant preparation procedure gave the high survival rate was 80-92.5% from removed 2-4 coleoptiles of axillary bud explant. This result suggests using axillary bud explant dissection and treating with drought stress could escape contamination from the interior contaminants. At the same procedure, more than 90% of survival rate was observed in axillary bud explants for successfully established of primary aseptic culture between tested cultivars（D. cv. Rudolph Roehrs and D. cv. Jupiter）. In addition, the influence of culture methods and TDZ concentration on shoot multiplication were investigated. For shoot multiplication, concentration of 1, 5, and 10mg/L TDZ in the liquid culture were found that shoot yields have a significant effect achieved 5-6 folds. While, 2-nodal explant was cultured in liquid medium only obtained 2 folds of multiplication rate, but compared to solid culture, the regenerated shoots were more rapid growth and uniformity during subculture. This result suggests that liquid culture can significantly enhance axillary shoot proliferation from stock stem explants and 2-nodal explants for mass multiplication in Dieffenbachia seedlings.
A rapid and efficient procedure is described for mass multiplication of microshoots using temporary immersion system (TIS) in dieffenbachia (Dieffenbachia ‘Starshine’). Multiple shoot proliferation was induced from 2-nodal and stock-stem explants on Murashige and Skoog (MS) media supplemented with 1 mg/L NAA and 0.1-5.0 mg/L thidiazuron (TDZ) or BA. Especially, the 2-nodal or stock-stem explants cultured under TIS on 5mg/L TDZ enriched medium had the highest shoot yield which was 24.5 and 59.2, respectively, the multiplication rate increased by 3.3-8 folds, over that obtained from conventional solid support systems. Therefore, the scheme for the rapid microshoot propagation of dieffenbachia using temporary immersion system had commercial efficiency. In addition, elongated multiple shoots after acclimatization, taken from the temporary immersion system, pretreated with 500mg/dm3 NAA then were cut and planted in plug with soilless mix under mist propagation condition to prevent desiccation. Survival was investigated after 30 days, microshoots taller than 3.0㎝ had the highest survival rate, which achieved 80-100%. Moreover, the shoot cluster was segmented into 1/4 or 1/2 section as propagules; the survival rate was 83.3-100%. In this production scheme, shoot cluster could be established a system of superior stock-plant for use in dieffenbachia cuttage.
A method for the somatic embryogenesis and subsequent plant regeneration for Dieffenbachia ‘Tiki’ hybrids was described. Male inflorescence explants isolated from spadix of flowering plants cultured in vitro, formed embryogenic calli on surfaces of inflorescence axis within eight weeks of culture on full-strength Murashiage and Skoog (MS) medium with 4-6 mg/L 2,4-D. Somatic embryogenesis and embryoid were induced using a modified half-strength MS combination of 2 % sucrose with 1 % glucose, 0.18 % Gelrite for the basal medium, supplemented with 4.0 to 5.1 mg/L 2,4-dichlorophenoxyacetic acid (2,4-D) and 0.5 mg/L Kinetin. Male inflorescence explants transfer to modified half-strength MS basal medium with 0.5mg/L Kinetin and 4mg/L 2,4-D（or 2-4mg/L Dicamba）resulted in somatic embryogenesis at frequencies of 45-72.1% with an average of 13.3-22.2 somatic embryos per responding explant. Furthermore, at the same culture condition, male inflorescence explants were cultured onto half-strength MS basal medium with 4mg/L 2,4-D and 0.5-1.0mg/L Thidiazuron（TDZ） for embryogenesis. TDZ was found to increase the somatic embryogenesis frequencies to 100% with an average of 32.1-38.2 somatic embryos per responding explant. At the same condition, a few somatic embryos developed into complete plantlets. In this study, we are the first to report somatic embryogenesis in Dieffenbachia ‘Tiki’ and the conversion of somatic embryos to greenhouse-established plant.
A regeneration system for improving the efficiency of somatic embryogenesis and emblings recovery from male inflorescences and somatic embryos of Dieffenbachia ‘Tiki’ were described. The highest percentage (100%) of primary somatic embryogenesis were achieved, and the highest yield of mature primary embryos were 75.5 on the half strength modified Murashige and Skoog（MS）medium with 2mg/L Dicamba and 0.5mg/L TDZ from male inflorescence explants. The results indicated that combination of Dicamba (2mg/L) and TDZ (0.5mg/L) significantly promoted high-efficiency multiplication of mature primary embryos in Dieffenbachia ‘Tiki’. Furthermore, the mature embryos of primary somatic embryogenesis were used as initial secondary explants for the induction of repetitive somatic embryogenesis. On the same culture medium supplemented with 2mg/L Dicamba and 0.5-1.0mg/L TDZ, the highest frequency (100%) of secondary embryo formation was obtained after 8-week subculture, and the highest number of mature secondary embryos per explant achieved 33.0-34.3 after 13-week subculture. Consequently, the same medium compositions were suitable for efficient repetitive somatic embryogenesis and multiplication of secondary embryos. In addition, emblings conversion of mature secondary embryos was investigated. The green secondary embryos of 4-5mm in size and the age of 8-10 weeks were used as explants, and cultured on half strength modified MS supplemented with 150mL/L coconut milk for somatic embryo conversion. The highest converted frequency of somatic embryo were 73.3%, and the number of converted emblings per explant reached 7.8 at 0.5% glucose and 1.0mg/L TDZ combination. Plantlets conversion from embryo was successfully acclimatized to greenhouse conditions. This technique could have significant industry application in dieffenbachia micropropagation, based on it has high efficiency of somatic embryo formation and high level of plant recovery.

並列關鍵字

Dieffenbachia ； micropropagation ； axillary bud formation ； somatic embryogenesis

參考文獻

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