臺灣博碩士論文加值系統

取青脆枝(Nothapodytes foetida)無菌實生苗的葉片、下胚軸為培植體，培養在MS basal salts + 0.5 mg/l BA + 0.5 mg/l 2,4-D建立癒合組織。將葉片癒合組織轉移至MS basal salts + 0.1 mg/l picloram再生不定根，並含有0.059% DW CPT。實生苗頂芽再生植株的根段，培養在MS basal salts + 1.0 mg/l BA + 0.1 mg/l 2,4-D誘導得不定根，含有0.0156% DW CPT，每30天的繼代培養可得到鮮重增加1.67倍。
葉片、下胚軸誘導的癒合組織及根段培植體培養在MS basal salts + 0.5 mg/l TDZ可間接再生不定芽，不定芽經BA誘導芽體抽長，再以IBA誘導發根，得到小植株，循序馴化，可出瓶培養。
取實生苗頂芽再生植株的葉片，經MS basal salts + 1 mg/l BA + 2 mg/l 2,4-D培養5個月，可得到胚性癒合組織。胚性癒合組織轉移到MS basal salt medium不添加生長調節劑，培養10週後開始有體胚發生，少數體胚可正常發育成植株，但多數體胚形態不正常。

Callus were induced from leaf and hypocotyl explants of sterile seedlings of Nothapodytes foetida cultured on MS basal salts + 0.5 mg/l BA + 0.5 mg/l 2,4-D. Adventitious roots regenerated from leaf-derived callus on MS basal salts + 0.1 mg/l picloram, and accumulated 0.059% DW CPT. Adventitious roots induced from root segments on MS basal salts + 1.0 mg/l BA + 0.1 mg/l 2,4-D, accumulated 0.0156% DW CPT after 30 days culture, and biomass increased 1.67 fold by subculture routinely.
Adventitious buds formed directly from root explants and indirectly from leaf and hypocotyl derived calli on MS basal salts + 0.5 mg/l TDZ. Adventitious buds elongated on MS basal medium supplemented with BA, and rooting by subculture on medium contained IBA. After acclimation, plantlets survived after transferred to soil.
Embryognenic callus induced from leaf explant of regenerants cultured on MS basal salts + 1 mg/l BA + 2 mg/l 2,4-D after 5 months culture. Then transferred to MS medium without PGRs, after 10 weeks culture, somatic embryogenesis was observed. Only few somatic embryos developed into plantlets, the others were morphologically abnormal.

ABLE OF CONTENTS
Abstract (in English)………………………………………………………i
Abstract (in Chinese)…………………………….………………………ii
Acknowledgments……………………………….………………………….iii
Table of contents..………………………………….……………………iv
List of Tables…………………………………….………………………viii
List of Figures………………………………….……………………………x
Abbreviations…………………………………..…………………….……xv
Chapter 1 Introduction..………………………….………………………1
1.1 Nothapodytes foetida..……………………….………………………1
1.2 Camptothecin..………………………………….………………………2
1.3 Studies correlated CPT with in vitro culture…………………5
1.3.1 Producing CPT through in vitro culture……….………….…5
1.3.2 Plant regeneration…………….…………….………………...10
Chapter 2 Methods and materials………………………….……………16
2.1 Materials.....…………………………………………………………16
2.2 The callus induction….………………….………………………..16
2.2.1 Establishment of sterile seedings and calli induction……16
2.2.2 Callus induction from immature inflorescence…………..….19
2.2.3 Callus induction from root explants…………..………………21
2.3 Maintenance of calli and CPT content of cells…………………21
2.3.1 Leave and hypocotyls explants.……...…………………………21
2.3.2 Inflorescence explants………………………….…………………27
2.3.3 Root explants.…………………..………………….………………27
2.4 Plant regeneration………….…………………………………………28
2.4.1 Organogenesis…………………………………………………………28
2.4.2 Embryogenic callus induction and embryogenesis ……………28
2.5 The harden of plant..…………………………………………………31
2.6 The CPT production of plant in field……………………….……32
2.7 The extraction of CPT………………………………..…..…………32
2.8 Analysis of CPT…………………………………….…………….……33
2.9 Standard of CPT…………………………………………………………33
2.10 The morphology observation and photograph……….……………33
2.11 Statistical analysis……………………………….…………...…33
Chapter 3 Results……………………………………………………………34
3.1 Callus induction and the CPT production of cells……………34
3.1.1 Establishment of sterile plantlet………………………………34
3.1.2 Hypocotyl explants…………………………………………………34
3.1.3 Leave explants.………………………………………………………41
3.1.4 Inflorescence explants………………………………………………46
3.1.5 Root explants…………………………………………………………50
3.2 Plantlet regeneration……………….……….………………………55
3.2.1 Organogenesis…………………………………………………………55
3.2.2 Induction of embryogenic callus and somatic embryogenesis………59
3.3 The harden of plantlet…………………………………………………66
3.4 The CPT content of plant in field …………….…………….……70
Chapter 4 Disscusion…………………………………………………………72
4.1 Callus induction and the CPT production of cells………………72
4.1.1 Establishment of sterile plantlet…………….…………………72
4.1.2 Callus induction from hypocotyls and leave..…….….………72
4.1.3 Inflorescence explants………………………………………………75
4.1.4 Root explants……………………………………………………………75
4.2 Plantlet regeneration…………………………….…………………76
4.2.1 Organogenesis………………………………....………………76
4.2.2 Embyogenic callus induction and embryogenesis…………77
4.3 The harden of plantlet……………………………………..………79
4.4 The CPT content of plant in field…………………….…………79
Chapter 5 Conclusions………………………………….………………………81
Chapter 6 References……………………………………………………………83

List of Tables
Table 1 CPTs content in tissue and organs…………….…………..……13, 14
Table 2 The basal compositions of MS (Murashige and Skoog, 1962)……18
Table 3 Composition of media for callus culture of flower stalk explants of Nothapodytes foetida……………….…………………….………………………22
Table 4 Composition of media for callus culture of root explants of Nothapodytes foetida……………………….……….……………………………23
Table 5 Composition of media for callus culture of hypocotyl explants of Nothapodytes foetida……………………………….….….………24
Table 6 Composition of media for callus culture of leaf explant of Nothapodytes foetida…………………...………….….…………...25
Table 7 Composition of shoot elongation for adventitious shoots induced from TDZ-induced nodular callus of Nothapodytes foetida…….....29
Table 8 Composition of rooting of adventitious shoots induced from TDZ-induced nodular callus of Nothapodytes foetida……..……....30
Table 9 Effect of media and culture conditions on biomass increase and CPT content of calli of hypocotyl explants of Nothapodytes foetida……………………………………………………….……..40
Table 10 Effect of media and culture conditions on biomass increase and CPT content of calli of leaf explants of Nothapodytes foetida……………………………………………………..……….48
Table 11 Effect of media and culture conditions on biomass increase and CPT content of calli of flower stalk explants of Nothapodytes foetida…………………………………………………….………..51
Table 12 Effect of media and culture conditions on biomass increase and CPT content of calli of root explants of Nothapodytes foetida..…………………………………………….……………....54
Table 13 Effect of BA on elongation of stunted shoot induced from TDZ- cultured nodular callus of Nothapodytes foetida cultured for 6 wk
………………………….. ………………………….……………...57
Table 14 Effect of IBA on rooting of 1-3 cm shoots induced from TDZ- cultured nodular callus of Nothapodytes foetida for cultured 5 wk
………………………………………………….………………….58
Table 15 Biomass increase of embryogenic callus induced from leaf of Nothapodytes foetida on MS + 1 mg/l BA + 2 mg/l 2,4-D solid or liquid media………………………………………....…………....61
Table 16 Effect of BA on conversion of somatic embryos induced from leaf of Nothapodytes foetida cultured for 2 months………………….65
List of Figures
Fig. 1 Chemical structure of camptothecin-related alkaloids. (Yamazaki et al., 2003).……………………...…………………………...........……...…3
Fig. 2 The predicted biosynthetic pathway of camptothecin in Ophiorrhiza pumila. Plausible biogenetic intermediates are put in parentheses. (Yamazaki et al., 2003)……………………………………..……..…..3
Fig. 3 Nothapodytes foetida…………………….………………..……...….17
Fig. 4 Immature flowers of Nothapodytes foetida…….….………….……..20
Fig. 5 The regenerats from shoot tip of seedling. (a) Callus induced from the base of shoot on MS medium + BA, and (b) plantlet. Bars= 1 cm….35
Fig. 6 Calli induced from hypocotyl explant of Nothapodytes foetida on MS + 0.5 mg/l BA + 0.5 mg/l 2,4-D, (a) white creamy callus induced on explant, (b) white creamy and compact green callus, and (c) nodular callus. Bars= 0.25 cm………………………………………..……....36
Fig. 7 Callus induced from hypocotyls of Nothapodytes foetida cultured on different composition of media and illuminations. (a) MS + 0.5 mg/l BA + 0.5 mg/l 2,4-D, (b) MS + 0.5 mg/l BA + 0.5 mg/l 2,4-D in dark (c) MS + 0.5 mg/l BA + 0.5 mg/l 2,4-D + 250 mg/l CH (d) MS + 0.5 mg/l BA + 0.5 mg/l 2,4-D + 3% coconut water. Bars = 1 cm.……..38
Fig. 8 (a) Nodular callus with shoot primordia (arrow), (b) adventitious shoots (arrow) and roots induced from hypocotyl explant of Nothapodytes foetida cultured on MS + 0.5 mg/l TDZ. Bars= 1 cm.……………………………………………….…………………39
Fig. 9 Calli induced from leaf explant of Nothapodytes foetida on MS + 0.5 mg/l BA+ 0.5 mg/l 2,4-D showed (a) green nodular callus with white creamy callus, bar represented 1 cm, and (b) green compact and whit friable callus, bar = 0.5 cm. ………………….………….42
Fig.10 Callus induced from leaf explant of Nothapodytes foetida cultured on MS + 0.5 mg/l BA + 0.5 mg/l 2,4-D for several subcultures, bar =1cm………………………………………….……………………43
Fig.11 (a)Calli induced from leaf explant of Nothapodytes foetida cultured on MS + 0.5 mg/l BA + 0.5 mg/l 2,4-D + 3% coconut water proliferated the clove-like structures (arrow), (b) after several subcultures some calli lost the clove-like structure and become fragile. Bars= 1 cm…………………………………..……………..44
Fig.12 (a) Calli induced by leaf explant of Nothapodytes foetida cultured on MS + 0.5 mg/l BA + 0.5 mg/l 2,4-D + 5% coconut water proliferated clove-like structure (arrow), (b) after several subcultures on the same media, calli lost the clove-like structure. Bars = 1 cm…………...…45
Fig.13 Adventitious roots regenerated from calli of leaf explant of Nothapodyte foetida cultured on MS + 0.1 mg/l picloram. Bar =1 cm…………………………………………….…………………….47
Fig.14 Calli of leaf explant of Nothapodyte foetida cultured on MS + 0.5 mg/l TDZ. Bar = 1 cm…………………………….………………..47
Fig. 15 Callus induced from flower stalk explants of Nothapodytes foetida on (a) MS + 0.5 mg/l 2,4-D (b) MS + 0.5 mg/l BA + 1 mg/l 2,4-D and (c) MS + 1 mg/l BA + 2 mg/l 2,4-D. Bars= 0.5 cm…...……………….49
Fig. 16 Root explant of Nothapodytes foetida cultured on MS + 0.5 mg/l TDZ. (a) In initial culture, TDZ cause root segment elongation and swelling, bar= 0.5 cm; (b) after subcultures, nodular callus, adventitious roots, and shoot primordia formed on root explants. Bar=1cm………………………………………….………………...52
Fig. 17 (a) Adventitious roots and creamy callus induced from root explants of Nothapodytes foetida on MS + 1.0 mg/l BA + 0.1 mg/l 2,4-D + 0.84 mg/l NAA. (b) After excises and subcultures, the adventitious roots may lost the original morphology and formed green compact callus. Bars= 1 cm……………………………………………..……53
Fig.18 Shoots induced from TDZ-induced nodular callus of Nothapodytes foetida. (a) Multiple shoots induced from hypocotyl explants. (b) Normal elongated shoot induced from hypocotyl explants, and (c) rooting shoot induced by IBA. Bars = 1 cm………….…………….56
Fig.19 Callus induced from leaf explants of in vitro plantlets of Nothapodytes foetida on MS + 1.0 mg/l BA + 2.0 mg/l 2,4-D. (a) Green compact callus, bar=0.5 cm; and (b) white friable callus regenerated on brownish leaf explant. Bar=1 cm…………….…….60
Fig.20 Callus induced from leaf explants of seedling of Nothapodytes foetida on MS + 0.5 mg/l BA + 0.5 mg/l 2,4-D transferred to MS + 1.0 mg/l BA + 2.0 mg/l 2,4-D regenerated white friable callus. Bar =1 cm....62
Fig.21 (a) Somatic embryogenesis from leaf-derived callus of Nothapodytes foetida subcultured on MS salt medium without PGRs, and (b) somatic embryos at different stages. Bar =1mm…………………....63
Fig.22 (a) Developing somatic embryo showed shoot apex and root (bar = 0.5 cm). (b) A somatic embryo-derived plantlet (bar =1.0 cm)….....64
Fig.23 Morphologic abnormalities of leaf-derived somatic embryos of Nothapodytes foetida. (a) Lack of cotyledon and apical meristem, (b) (c) 2 embryo fused, (d) lack of root. Bar = 0.5 cm………...……….67
Fig.24 (a) Globular embryo (bar=0.2 mm) and (b) cellular division (bar=0.025 mm) derived from suspension of leaf Nothapodytes foetida………………………………………………….…………...68
Fig.25 The somatic embryos developed in suspention culture of embryogenic cullus of Nothapodytes foetida for (a) 2 months and (b) 4 months, bar = 1 cm…………………………………………………………..…..69
Fig.26 Plant potted in vermiculite: perlite: peat moss (1:1:1)…….………..70

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