臺灣博碩士論文加值系統

二倍體石蒜胚乳培養參試種有紅花石蒜（Lycoris radiata）、金花石蒜（L. aurea）、換錦石蒜（L. sprengeri）及紅花石蒜×換錦石蒜（L. radiata ×L. sprengeri）等。本研究目的主要探討胚乳組織發育與再生能力之關係，及影響其擬胚發生和器官發生之因子，並期由胚乳體外培養再生三倍體、多倍體或非整倍體植株。
1. 胚乳發育期：二倍體石蒜種子由授粉至成熟所需生育日數約3個月。胚乳組織生長發育可分為核型期（開花至授粉後10天）、細胞型期（授粉後10-35天）、固化期（授粉後45-60天）及成熟期（授粉後60天之後）等四個階段。於核型期之前或固化期之後進行胚乳培養難以誘導再生。細胞型期胚乳細胞分裂活性高，受外加不同生長調節劑影響，具多重形態發生潛力。
2. 不同種石蒜及其雜交胚乳培養擬胚及不定芽發生：胚乳組織再生潛力依序為A群種（紅花石蒜及換錦石蒜）、 M-T群種（金花石蒜）、M-T-A群種（紅花石蒜 ×金花石蒜及金花石蒜 ×紅花石蒜）。不同種及授粉組合適合誘導癒合組織發生的外加生長調節劑濃度及種類有所差異。胚乳衍生癒合組織接種於添加NAA（0.1、0.5或3 mg/l）及10 mg/l BA之MS培養基經5-12個月（隨授粉組合而有所不同）可進一步分化擬胚或不定芽。胚乳衍生擬胚及不定芽於添加NAA（0.1或1.0 mg/l）及BA（0.1或1.0 mg/l）之MS培養基可誘導擬胚發芽、不定芽發根、瓶內結球及抽葉。
3. 形態發生解剖觀察：細胞型期胚乳組成細胞成異質性，胚乳培養於再生培養基，胚乳衍生癒合組織細胞分裂形成擬分生帶，外分生帶之原表皮或次原表皮細胞可再分化擬胚或不定芽。
4. 細胞學檢定：紅花石蒜及換錦石蒜胚乳可再生小植株。紅花石蒜胚乳再生小植株染色體數為3n=33A，換錦石蒜胚乳再生小植株染色體數為3n=29-34。金花石蒜、紅花石蒜 ×換錦石蒜胚乳及紅花石蒜 ×金花石蒜雜交胚乳僅有不定芽及擬胚發生，金花石蒜 ×紅花石蒜雜交胚乳僅逆分化癒合組織。經檢驗其衍生癒合組織細胞得知，以非整倍體及多倍體細胞為多數，且有異常染色體及細胞分裂行為。此外，同一植株體細胞染色體亦呈現非均一鑲嵌現象。

The purpose of this research is intending to study the morphogenic competence including embryogenesis and organogenesis in the endosperm culture of Lycoris radiata, L. aurea, L. sprengeri, L. radiata ×L. sprengeri,and reciprocal between L. aurea and L. radiata.
1. Endosperm development：The development of endosperm could be classified into nuclear、cellular、solid and mature stage. The developmental time table requires 2.5-3 months. Embryoids or adventitious buds were difficult to regenerate from nuclear、solid, and mature endosperm of the species studied. Cellular endosperm has high mitotic activity and multiple morphogenic competence.
2. Organogenesis and embryogenesis in the endosperm culture of Lycoris taxa：
A karyotype groups (L. radiata and L. sprengeri) have higher morphogenic competence than M-T(L. aurea) or M-T-A (reciprocal between L. aurea and L. radiata) karyotype groups. Embryoids or adventitious buds from callus were induced by transferring to MS basal medium supplemented with NAA(0.1, 0.5 or 3 mg/l) and 10 mg/l BA after 5-12 months subculture(subculture period varies with species). Endosperm-derived embryoids or adventitious buds were transferred to MS basal medium supplemented with NAA(0.1 or1.0 mg/l) and BA(0.1 or 1.0 mg/l) as to germination, rooting, in vitro bulbing and leaf emergence.
3. Anatomical observation of morphogenesis：Endosperm-derived callus redifferentiated meristematic zone . Embryoids or adventitious buds were regenerated from the newly formed proepidermis cells.
4. Cytological examination：The somatic chromosomes of endosperm-derived plantlets of L. radiata and L. sprengeri were examined to be 3n=33A and 3n=29-34 respectively. Endosperm-derived callus of L. aurea, L. radiata ×L. sprengeri, and reciprocal between L. aurea and L. radiata displayed variable chromosome number. Additionally, irregular cell division,aneuploid and polyploid cytochimeras were also observed.

內容目次
一、前言（Introduction）1
二、前人研究（Review）3
A. 植物多倍體化途徑3
（一）有性多倍體化3
（二）無性多倍體化4
1.人工染色體加倍4
2.體細胞雜交4
（三）有性兼無性多倍體化5
B. 胚乳起源及發育形態特性5
（一）胚乳起源5
（二）胚乳發育類型6
1.核型胚乳（free nuclear endosperm）6
2.細胞型胚乳（cellular endosperm）7
3.沼生目型胚乳（helobial type endosperm）7
C. 胚乳組織培養8
(一) 胚乳培養再生途徑8
1.擬胚發生（embryogenesis）8
a.直接擬胚發生(direct embryogenesis)8
b.間接擬胚發生(indirect embryogenesis)9
c.中間型擬胚發生(intermediate embryogenesis)9
d.重複擬胚發生(repetitive embryogenesis)9
2.器官發生（organogenesis）10
a.不定芽發生（caulogenesis）10
b.不定根發生（rhizogenesis）10
（二）影響胚乳組織再生之因子11
1.基因型11
2.成熟度11
3.胚因素12
4.生長調節物質12
5.環境因子13
（三）胚乳培養組織學和細胞學研究13
三、材料與方法（Materials and methods）15
（一）參試材料及培植體15
（二）胚乳組織發育形態及解剖觀察15
1. 解剖顯微鏡形態觀察15
2. 石蠟切片觀察16
（三）影響胚乳組織培養再生因子16
1. 不同種及雜交胚乳再生比較16
2. 胚乳發育期17
3. 胚因素17
4. 植物生長調節劑17
（1）癒合組織誘導17
（2）不定芽及體胚誘導17
（3）小植株建立18
（四）胚乳培養再生途徑及解剖形態觀察18
（五）胚乳衍生癒合組織及再生株細胞學檢查18
四、結果（Results）19
（一）二倍體石蒜結實、種子及胚乳組織發育特性及解剖形態19
（二）影響胚乳組織培養再生因子21
1.種皮及胚因素21
2.胚乳發育期24
3.生長素（NAA, 2,4-D）與BA或GA之組合對不同石蒜種自交及雜交胚乳再生潛力之關係25
（三）胚乳培養再生途徑及解剖形態觀察27
（四）二倍體石蒜胚乳再生小植株建立27
（五）石蒜胚乳再生植株及其衍生癒合組織細胞學檢查28
1.石蒜胚乳再生植株細胞學檢定28
(1)紅花石蒜28
(2)換錦石蒜28
2.石蒜胚乳衍生癒合組織細胞學檢定28
(1)金花石蒜28
(2)金花石蒜 ×紅花石蒜29
(3)紅花石蒜 ×金花石蒜29
(4)紅花石蒜 ×換錦石蒜29
五、討論（Discussion）57
（一）二倍體石蒜胚乳組織發育時期與再生潛力之關係57
（二）石蒜胚乳組織再生途徑影響因子及形態發育解剖特性61
1.石蒜胚乳組織再生途徑影響因子61
2.胚乳培養形態發育解剖特性62
（三）不同種石蒜種內自交或種間雜交胚乳組織再生植株及其衍生癒合組織細胞學表現64
六、摘要69
七、Summary71
八、參考文獻（Reference）72
表目次
表1. 金花石蒜種皮、胚、胚乳及珠心組織發育相關特性23
Table 1. The development and growth of seed coat,embryo,endosperm and nucellus in Lycoris aurea seed23
表2. 種皮及胚對不同授粉後天數石蒜胚乳癒合組織發生之影響24
Table 2. The effect of seed coat and embryo on callusing in endosperm culture of Lycoris taxa at different development stage24
表3. 生長素（NAA,2,4-D）與BA或GA之組合對不同種石蒜及雜交胚乳組織衍生癒合組織發生之影響31
Table 3. The effect of NAA and BA on callusing in Lycoris taxa endosperm31
表4. NAA與BA組合對不同種石蒜自交及雜交胚乳培養癒合組織形成及再生時間之影響31
Table 4. The effect of NAA and BA on regeneration in Lycoris taxa endosperm31
表5. 不同種石蒜自交及雜交胚乳衍生小植株及癒合組織觀察之細胞染色體數目32
Table 5. The observed and expected chromosome number in the endosperm-derived plantlets and callus of selfing34
附錄1.植物多倍體化方法之比較78
Appendix 1.The methods of polyploidization.78
圖目次
圖1. 金花石蒜、紅花石蒜、換錦石蒜及紅花石蒜 ×換錦石蒜開花、結實及種子生長發育特性33
Fig.1. The anthesis,fruiting,and seed development of Lycoris aurea,L. radiata,L. sprengeri,L. radiata ×L. sprengeri.33
圖2-1. 金花石蒜胚珠及胚乳發育解剖形態35
Fig.2-1. Anatomical features of ovule and developing endosperm35
圖2-2. 金花石蒜胚珠及細胞化胚乳發育解剖形態37
Fig.2-2. Anatomical features of ovule and developing cellular stage endosperm37
圖3. 紅花石蒜胚乳培養再生擬胚及小植株建立39
Fig.3. The regeneration of endosperm-derived embryoids and plantlets of Lycoris radiata39
圖4. 換錦石蒜及紅花石蒜 ×換錦石蒜胚乳培養再生擬胚、不定芽及小鱗莖建立41
Fig.4. The regeneration of endosperm-derived embryoids 、adventitious buds and bulblets of Lycoris sprengeri and L. radiata ×L. sprengeri41
圖5. 金花石蒜胚乳及紅花石蒜 ×金花石蒜雜交胚乳培養再生擬胚及不定芽43
Fig.5. The regeneration of endosperm-derived embryoids and adventitious buds of Lycoris aurea and L. radiata ×L. aurea43
圖6. 紅花石蒜胚乳培養再生擬胚及不定芽形態發生解剖特性45
Fig.6. The anatomical features and morphogenesis of endosperm-derived embryoids and adventitious buds in Lycoris radiata endosperm culture45
圖7. 紅花石蒜及換錦石蒜胚乳培養再生植株細胞學檢查47
Fig.7. The cytological examination in endosperm-derived plantlets of Lycoris radiata and L. sprengeri47
圖8. 金花石蒜胚乳衍生癒合組織細胞學檢查49
Fig.8. The cytological examination in endosperm-derived callus of Lycoris aurea49
圖9. 金花石蒜 ×紅花石蒜及紅花石蒜 ×金花石蒜雜交胚乳衍生癒合組織細胞學檢查51
Fig.9. The cytological examination in endosperm-derived callus of L. radiata and L. aurea reciprocal interspecific hybrids51
圖10. 紅花石蒜 ×換錦石蒜胚乳衍生癒合組織細胞學檢查53
Fig.10. The cytological examination in endosperm-derived callus of L. radiata ×L. sprengeri hybrid endosperm culture53
圖11. 石蒜胚乳衍生小植株及癒合組織其異常染色體行為及細胞分裂情形55
Fig.11. Irregular chromosomal behavior and mitosis in endosperm- derived plantlets and callus of Lycoris taxa55

毋錫金、劉淑瓊、周月坤、錢南芬、張鵬、謝鴻勛、張福壽、閻振龍. 1977. 蘋果胚乳癒傷組織的誘導和植株分化. 中國科學 4:355-359.
阮明淑、吳奇璋、許圳塗. 1997a. 石蒜種源及育種概況（下）. 農業世界 172：82-84.
阮明淑、梁素秋、許圳塗. 1997b. 新興球根花卉石蒜之生育及生長週期特性.農業世界：35-40.
阮明淑. 2000. 金花石蒜及相關種遺傳歧異性分析及核型重塑之研究. 台大園藝學研究所博士論文. pp8-16.
李淑真. 1997. 百香果胚乳組織培養不定芽及體胚誘導之研究. 台大園藝學研究所碩士論文. 105p.
林定勇、李哖. 1993. 石蒜屬球根花卉之分類、型態、生長與開花. 中國園藝 39：67-72.
胡適宜. 1990. 被子植物胚胎學. 曉園出版社. 台北. p118-129.
高佑靈. 1993. 玉米胚乳發育初期細胞分裂素與生長素的分佈變化. 台大農藝學研究所碩士論文. pp1-15.
陳振光. 1987. 果樹組織培養. 上海科學技術出版社. 上海. p139-148.
張本雲. 1994. 中國作物遺傳資源. 中國農業學會遺傳資源學會編. 中國農業出版社. 北京. p1172-1177.
蔡淑華. 1995. 植物組織切片技術綱要. 茂昌股份有限公司. 台北. 72pp.
劉秋芳. 1999. 二倍體香蕉胚乳培養器官再生及體胚發生之研究. 台大園藝學研究所碩士論文. 70p.
Adiwilaga, K. D., and C. R. Brown. 1991. Use of 2n pollen-producing triploid hybrids to introduce tetraploid Mexican wild species germ plasm to cultivated tetraploid potato gene pool. Theor. Appl. Genet. 81:645-652.
Averett, J. E. 1980. Polyploidy in plant taxa. Lewis W. H. ed. Polyploidy,biological relevance. New York. Plenum Press. p269-273.
Bhaskaran, S., and R. H. Smith. 1990. Regeneration in cereal tissue culture：A review. Crop Sci. 30：21-30.
Bharathan, G. 1996. Reproductive development and nuclear DNA content in angiosperms. Amer. J. Bot. 83:440-451.
Bhojwani, S. S. 1971. Morphogenetics studies on culture mature endosperm of Croton bonplandianum Baill. New Phytol. 70:761-766.
Bretagnolle, F., and J. D. Thompson. 1995. Gametes with the somatic chromosome number: mechanisms of their formation and role in the evolution of autopolyploid plants. New Phytol. 129:1-22.
Brooker. R. J. 1999. Genetics analysis and principles. Addison Wesley Longman, Inc. p211-226.
Brown, R. C., B. E. Lemmon, and O. A. Olsen. 1994. Endosperm development in barley:Microtubule involvement in the morphogenetic pathway. Plant cell 6:1241-1252.
Brown, R. C., B. E. Lemmon, and O. A. Olsen. 1996. Development of the endosperm in rice (Oryza sativa L.):Cellularization. J. Plant Res. 109:301-313.
Cantoni, L., G. Berardi, and P. Rosati. 1993. Callus induction from endosperm culture of blackberry. Acta Hort. 352:145-149.
Ceniza, M. S., S. Ueda, and Y. Sugimura. 1992. In vitro culture of coconut endosperm:callus induction and its fatty acids. Plant Cell Reports 11:546-549.
Clark, F. J., and F. C. Copeland. 1940. Chromosome aberrations in the endosperm of maize. Amer. J. Bot. 27:247-251.
Edward, C. Y., and J. C. Michael. 1987. Cellular endosperm formation in Phaseolus vulgaris.Ⅰ. Light and scanning electron microscopy. Can. J. Bot. 66:1209-1216.
Friedman, W. E. 1994. The evolution of embryogeny in seed plants and the developmental origin and early history of endosperm. Amer. J. Bot. 81:1468-1486.
Garg, L., N. N. Bhandari, V. R. Ani, and S. S. Bhojwani. 1996. Somatic embryogenesis and regeneration of triploid plants in endosperm cultures of Acacia nilotica. Plant Cell Rep. 15:855-858.
Gmitter, F. G., Jr. X. B. Ling, and X. X. Deng. 1990. Induction of triploid Citrus plants from endosperm calli in vitro. Theor. Appl. Genet. 80:785-790.
Hartmann, H. T., D. E. Kester, F. T. Davies Jr., and R. L. Geneve.1997. Plant propagation:principles and practices. Simon&Schuster/ A Viacom Company. 770pp.
Johri, B. M., and S. S. Bhojwani. 1965. Growth responses of mature endosperm in cultures. Nature 208:1345-1347.
Johri, B. M., P. S. Srivastava. 1973. Morphogenesis in endosperm culture. Z. Pflanzenphysiol 70:285-304.
Kurita, S. 1988. Variation and evolution in the karyotype of Lycoris, Amaryllidaceae Ⅶ. Modes of Karyotype alteration within species and probable trend of karyotype evolution in the genus. Cytologia 53:323-335.
La Rue C. D. 1949. Growth of the endosperm of maize. Amer. J. Bot. 36:798.
Lee, K. H. 1992. Plant phenolic compounds as cytotoxic antitumor agent. ACS-Sym-ser. Washington, D. C. Ameri. Chemi. Soci. 507:367-379.
Liu, C. M., and W. M. David. 1998. The titan mutants of Arabidopsis are disrupted in mitosis and cell cycle control during seed development. Plant J. 16:21-31.
Lopes, M. A., and B. A. Larkins. 1993. Endosperm origin, development, and function. Plant Cell 5:1383-1399.
Miller, S. S., L. A. A. Bowman, M. Gijzen, and B. L. A. Miki. 1999. Early development of the seed coat of soybean (Glycine max). Annu. Bot. 84:297-304.
Mohamed, M. E., R. G. T. Hicks, and D. Blakesley. 1996. Shoot regeneration from mature endosperm of Passiflora foetida. Plant Cell, Tissue and Organ Culture 46:161-164.
Mu, S. K. and S. Q. Liu. 1979. Studies on initiation of apple endosperm callus and variation of chromosomal ploidy of callus cell of endosperm. Acta. Bot. Sinica. 21:309-311.
Mu, S. K., L. G. Fraser, and C. F. Harvey. 1990. Initiation of callus and regeneration of plantlets from endosperm of Actinidia interspecific hybrids. Sci. Hort. 44:107-117.
Murashige, T., and F. S. Skoog. 1962. A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol. Plant. 15:473-497.
Nair, S., M. V. Shirgurkar, and A. F. Mascarenhas. 1986. Studies on endosperm culture of Annona squamosa Linn. Plant Cell Reports 5: 132-135.
Nag, K. K., and B. M. Johri. 1971. Morphogenetic studies on endosperm of some parasitic angiosperms. Phytomorphogy 21:202-218.
Norstog, K. J. 1956. Growth of rye-grass endosperm in vitro. Bot. Gaz. 117:253-259.
Olsen, Q. A., R. H. Potter, and R. Kalla. 1992. Histo-differentiation and molecular biology of developing cereal endosperm. Seed Sci. Res. 2:117-131.
Pennell, R., and C. Lamb. 1997. Programmed cell death in plants. Plant Cell 9:1157-1168.
Rangaswamy, N. S., and P. S. Rao. 1963. Experimental studies on Santalum album L.-Establishment of tissue culture of endosperm. Phytomorphology 13:450-454.
Salon, P. R., and E. D. Earle. 1998. Chromosome doubling and mode of reproduction of induced tetraploids of eastern gamagrass(Tripsacum dactyloides L.). Plant Cell Report 17:881-885.
Satsangi, A., and H. Y. MohanRam. 1965. A continuously growing tissue culture from the mature endosperm of Ricinus Communis. Phytomorphogy 15:26-30.
Sehgal, C. B., and S. Khurana. 1985. Morphogenesis and plant regeneration from cultured endosperm of Emblica officinalis Gaertn. Plant Cell Reports 4:236-266.
Siro, K. 1988. Variation and evolution in the karyotype of Lycoris, AmaryllidaceaeⅥ. Intrapopulational and/or intraspecific variation in the karyotype of L. sanguinea Max. var. kiushiana and L. sanguinea Max. var. koreana (Nakai) Koyama. Cytologia 53:307-321.
Stephen, J. 1979. Cytological causes of spontaneous fruit abortion in Haemanthus katherinae Baker. Cytologia 44:805-812.
Straus, J. 1954. Maize endosperm tissue grown in vitro.Ⅱ morphology and cytology. Amer. J. Bot. 41:833-839.
Straus, J., and C. D. LaRue. 1954. Maize endosperm tissue growth in vitro.ⅠCulture requirements. Amer. J. Bot. 41:833-839.
Tao, R., K. Ozawa, M. Tamura, and A. Sugiura. 1997. Dodecaploid plant regeneration from endosperm culture of persimmon(Diospyros kaki L.). Acta Hort. 436:119-128.
Tulecke, W., G. McGranahan, and H. Ahmadi. 1988. Regeneration by somatic embryogenesis of triploid plants from endosperm of walnut,Juglans regia L. cv manregian. Plant Cell Reports 7:301-304.
Tusa, N., J. W. Grosser, and F. G. Gmitter, Jr. 1990. Plant regeneration of ‘Valencia’sweet orange,’Femminello’ lemon, and the interspecific somatic hybrid following protoplast fusion. J. Amer. Soc. Hort. Sci. 115:1043-1046.
Wang, M., B. J. Oppedijk, X. Lu, B. V. Duijn, and R. A. Schilperoort. 1996. Apoptosis in barley aleurone during germination and its inhibition by abscisic acid. Plant Mol. Biol. 32:1125-1134.
Wang, M., B. J. Oppedijk, M. P. M. Caspers, G. E. M. Lamers, M. J. Boot, D. N. G. Geerlings, B. Bakhuizen, A. H. Meijer, and B. V. Duijn. 1998. Spatial and temporal regulation of DNA fragmentation in the aleurone of germinating barley. J. Exp. Bot. 49:1293-1301.
Webb, M. C., and B. E. S. Gunning. 1991. The microtubular cytoskeleton during development of the zygote,proembryo and free-nuclear endosperm in Arabidopsis thaliana (L.) Heynh. Planta 184:187-195.
Williams, E. G., and G. Maheswaran. 1986. Somatic embryogenesis:factors in fluencing coordinated behaviour of cells as an embryogenic group. Ann. Bot. 57:443-462.
Wolters, A. M. A., H. C. H. Schoenmakers, S. Kamstra, J. Van Eden, M. Koornneef, and J. H. DE Jong. 1994. Mitotic and meiotic irregularities in somatic hybrids of Lycopersicon esculentum and Solanum tuberosum. Genome 37:726-735.
Van Lammeren, A. A. M. 1988. Structure and function of the microtubular cytoskeleton during endosperm development in wheat:An immuno-fluorescent study. Protoplasma 146:18-27.
Xuhan, X., and A. A. M. Van Lammeren. 1994. Microtubular configurations during endosperm development in Phaseolus vulgaris. Can. J. Bot. 72:1489-1495.
Young, T. E., D. R. Gallie, and D. A. DeMason. 1997. Ethylene-mediated programmed cell death during maize endosperm development of wild-type and shrunken 2 genotypes. Plant Physiol. 115:737-751.
Young, T. E., and D. R. Gallie. 1999. Analysis of programmed cell death in wheat endosperm reveals differences in endosperm development between cereals. Plant Mol. Biol. 39:915-926.
Zhao, H., and N. Gu. 1983. Endosperm culture in pear. Handbook of Plant Cell Culture. Vol. 6. Perennial Crops. Eds Z. Chen, D. A. Evans, W. R. Sharp, P. V. Ammirato and M. R. Sondahl. McMgraw-Hill. New York. p274-277.