臺灣博碩士論文加值系統

甘藷（sweet potato；Ipomoea batatas（L）Lam.）為熱帶作物，由於具有不耐低溫之生理特性，故無法以冷馴化的方式，誘導其產生耐寒性與耐旱性。為了達到超低溫冷凍保存之目的，以0.3 M蔗糖進行前處理，藉由適度的滲透逆境，誘導植株產生耐寒性與耐旱性。
台農68品系甘藷，以株齡80天之植株為材料，進行0.3 M蔗糖前處理7天，配合LS處理60分鐘，PVS2處理55分鐘，超低溫冷凍保存後，可達到22.2 % 之存活率。台農57品系甘藷，以株齡40天之植株為材料，進行0.3 M蔗糖前處理3天，配合LS處理60分鐘，PVS2處理35至55分鐘，超低溫冷凍保存後，可達到87.5至100 % 之存活率。
為了解影響甘藷超低溫冷凍保存存活率之主要因素，將不同株齡之植株處理0.3 M蔗糖不同天數，進行相對含水量、滲透潛勢、可溶性醣類與可溶性蛋白質含量之測定。發現可溶性醣類含量為影響滲透潛勢變化之主要因素，而當可溶性醣類含量與滲透潛勢具有高相關時，超低溫冷凍保存存活率則較高。另外也發現，在滲透逆境下，滲透潛勢下降幅度愈大、下降速度愈快且愈持久時，超低溫冷凍保存存活率亦較高。藉由上述結果，推測0.3 M蔗糖處理1至7天，為較佳冷凍保存處理天數。亦推測年輕植株之莖頂較成熟植株更適合進行超低溫冷凍保存之處理。而在滲透逆境下，累積的可溶性醣類主要為雙醣且具足夠累積含量時，亦可達到較高之超低溫冷凍保存存活率。

Sweet potato（Ipomoea batatas（L）Lam.）is a tropical plant which is unable to be cold-hardened. To achieve cryopreservation of this plant, We have used osmotic stress to replace the treatment of low temperatures. Suitable osmotic stress induced by 0.3 M sucrose preculture is aimed at enhancing the tolerance to desiccation and freezing of this plant to be cryopreserved.
The shoot tips of sweet potato TN68（Ipomoea batatas（L）Lam. cv. Tainung No. 68）were excised from 80-day-old in vitro-grown plantlets and pretreated on solidified MS basal medium supplemented with 0.3 M sucrose for 7 days. The precultured shoot tips were treated with a loading solution（LS）for 60 minutes and then dehydrated with a vitrification solution（PVS2）for 55 minutes prior to a plunge into liquid nitrogen. The post-thaw survival rate of shoot tips was up to 22.2 % .
The shoot tips of sweet potato TN57（Ipomoea batatas（L）Lam. cv. Tainung No. 57）were excised from 40-day-old in vitro-grown plantlets and pretreated on solidified MS basal medium supplemented with 0.3 M sucrose for 3 days. The precultured shoot tips were treated with a loading solution（LS）for 60 minutes and then dehydrated with a vitrification solution（PVS2）for 35-75 minutes prior to a plunge into liquid nitrogen. The post-thaw survival rates of shoot tips was up to 87.5-100 % .
To understand the action of sucrose in conferring desiccation and freezing tolerance, we analyzed the hydric and biochemical modification in pretreated plantlets. The results indicated that the accumulation of soluble sugar was the main effect that leaded to the decrease of osmotic potential. When the correlation of soluble sugar content and osmotic potential was high, the survival rate of cryopreservation was also high. When the decrease of osmotic potential was large and more rapid and lasting, the survival of cryopreservation was also better. Based on the results shown above, the best pretreatment time was 1-7 days and the shoot tips from young plantlets was better than old plantlets for cryopreservation. What kinds of soluble sugars accumulate after osmotic stress is important. We found that the accumulation of disaccharide can make the survival of cryopreservation to be better.

目錄……………………………………………………I
表目錄…………………………………………………II
圖目錄…………………………………………………III
縮寫表…………………………………………………IV
摘要……………………………………………………1
Abstract………………………………………………2
一、前言………………………………………………3
二、前人研究…………………………………………5
三、材料與方法………………………………………10
四、結果………………………………………………16
五、討論………………………………………………47
六、未來展望…………………………………………54
七、參考文獻…………………………………………55
附錄……………………………………………………60

王志恭。1998。缺水處理對番木瓜葉片光能利用及抗氧化系統之影響。國立中興大學植物學研究所碩士論文。
宋龍瑛。2001。蔗糖及冷馴化處理對樹薯玻璃化溶液（PVS2）忍受度及生理代謝之影響。國立中興大學植物學研究所碩士論文。
李良。1963。甘藷。台灣雜糧作物品種圖說。台灣省政府農林廳。pp. 1-47
陳建山。1995。甘藷產業新展望。鄉間小路 21（4）：7-8
廖嘉信。1993。食用甘藷品種。嘉義農業試驗分所。嘉義。
廖嘉信。1995。塊根甘藷。鄉間小路 21（4）：12-13
Benson EE. ( 1994 ) Cryopreservation. In : Dixon RA. and Gonzales RA. ( ed. ) Plant cell culture - a practical approach ( second edition ). Oxford University, New York, pp.147-167
Bhatti MH., Percival T., Davey CDM., Henshaw GG. and Blakesley D. ( 1997 ) Cryopreservation of embryogenic tissue of a range of genotypes of sweet potato [ Ipomoea batatas ( L ) Lam. ] using an encapsulation protocol. Plant Cell Reports 16 : 802-806
Blakesley D., AI Mazrooei S. and Henshaw GG. ( 1995 ) Cryopreservation of embryogenic tissue of sweet potato ( Ipomoea batatas ) : use of sucrose and dehydration for cryoprotection. Plant Cell Reports 15 : 259-263
Blakesley D., AI Mazrooei S., Bhatti MH. and Henshaw GG. ( 1996 ) Cryoperservation of non-encapsulated embryogenic tissue of sweet potato ( Ipomoea batatas ) . Plant Cell Reports 15 : 873-876
Blakesley D., Percival T., Bhatti MH. and Henshaw GG. ( 1997 ) A simplified protocol for cryopreservation of embryogenic tissue of sweet potato [ Ipomoea batatas ( L ) Lam. ] utilising sucrose preculture only. Cryo-Letters 18 : 77-80
Charoensub R., Phansiri S., Sakai A. and Yongmanitchai W. ( 1999 ) Cryopreservation of cassava in vitro-grown shoot tips cooled to —196℃ by vitrification. Cryo-Letters 20 : 89-94
Chu C., Dai Z., Maurice SB. and Edward GE. ( 1990 ) Induction of crassulacean acid metabolism in the facultative halophyte Mesembryanthemum Cryssstallinum by abscisic acid. Plant Physiol. 93 : 1253-1260
Crowe JH., Carpenter JF., Crowe LM. and Anchordoguy TJ. ( 1990 ) Are freezing and dehydration similar stress vectors ? A comparsion of modes of interaction of stabilizing solutes with biomolecules. Cryobiology 27 : 219-231
Crowe JH., Crowe LM., Carpenter JF., Rudolph AS., Wistorm CA., Spargo BJ. And Anchordoguy TJ. ( 1989 ) Interactions of sugar with membranes. Biochimica et Biophysica Acta 947 : 367-384
Englemann F. ( 1991 ) In vitro conservation of tropical plant germplasm - a review. Euphytica 57 : 227-243
Gonzalez-Arnao MT., Ravelo MM., Villavicencio CU., Montero MM. and Englemann F. ( 1998 ) Cryopreservation of pineapple ( Ananas comosus ) apices. Cryo-Letters 19 : 375-382
Grospietsch M., Stodulkova E. and Zamecnik J. ( 1999 ) Effect of osmotic stress on the dehydration tolerance and cryopreservation of Solanum tuberosum shoot tips. Cryo-Letters 20 : 339-346
Hitmi A., Coudret A., Barthomeuf C. and Sallanon H. ( 1999 ) The role of sucrose in freezing tolerance in Chrysanthemum cinerariaefolium L. cell cultures. Cryo-Letters 20 : 45-54
Houssa P., Bernier G. and Kinet JM. ( 1991 ) Qualitative and quantitative analysis of carbohydrates in leaf exudate of the short-day plant, Xanthium strumarium L. during floral transition. J. Plant Physiol. 138 : 24-28
Kuo CG. ( 1990 ) Conservation and distribution of sweet potato germplasm. In : Dodds JH ( ed. ) In vitro methods for conservation of plant genetic resources. Chapman and Hall, London, pp.123-150
Luo J. and Reed BM. ( 1997 ) Abscisic acid-responsive protein, bovine serum albumin, and proline pretreatments improve recovery of in vitro currant shoot-tip meristems and callus cryopreservation by vitrification. Cryobiology 34 : 240-250
Matsumoto T., Sakai A. and Nako Y. ( 1998 ) A novel preculturing for enhancing the survival of in vitro-grown meristems of wasabi ( Wasabia japonica ) cooled to -196℃by vitrification. Cryo-Letters 19 : 27-36
Murashige T. and skoog F. ( 1962 ) A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol Plant 15 : 473-497
Oliver AE., Crowe LM. and Crowe JH. ( 1998 ) Methods for dehydration-tolerance : depression of the phase transition in dry membranes and carbohydrate vitrification. Seed Science Research 8 : 211-221
Pennycooke JC. and Towill LE. ( 2000 ) Cryopreservation of shoot tips from in vitro plants of sweet potato [ Ipomoea batatas ( L ) Lam. ] by vitrification. Plant Cell Reports 19 : 733-737
Pierce A. (1989 ) BCA protein assay reagent. Introductions Pierce Chemical Company. Printed in U.S.A.
Plessis P. and Steponkus PL. ( 1996 ) Cryopreservation of sweet potato shoot-tips by vitrification. Cryobiology 33 : 655-656
Pritchard HW., Grout BWW. and Short KC. ( 1986 ) Osmotic stress as a pregrowth procedure for cryopreservation. 2. Water relations and metabolic state of sycamore and soybean cell suspensions. Annals of Botany 57 : 371-378
Sakai AS., Kobayashi S. and Oiyama I. ( 1990 ) Cryopreservation of nucellar cells of navel orange ( Citrus sinensis Osb.var. brasiliensis Tanak ) by vitrification. Plant Cell Reports 9 : 30-33
Swan TW., O’Hare D., Gill RA. and Lynch PT. ( 1999 ) Influence of preculture conditions on the post-thaw recovery of suspension cultures of Jerusalem artichoke ( Helianthus tuberosus L. ). Cryo-Letters 20 : 325-336
Takagi H., Thinh NT., Islam OM., Senboku T. and Sakai A. ( 1997 ) Cryopreservation of in vitro-grown shoot tips of taro ( Colocasia esculenta ( L.) schott ) by vitrification. 1. Investigation of basic condition of the vitrification procedure. Plant Cell Reports 16 : 594-599
Takagi H. ( 1998 ) Recent developments in cryopreservation of shoot apices of tropical species. In : Engelmann F. and Takagi H. ( eds ) Cryopreservation of tropical plant germplasm. Tsukuba, Japan, pp.178-193
Tarczynski MC., Byrne DN. and Miller WB. ( 1992 ) High performance liquid chromatography analysis of carbohydrates of cotton-phloem sap and of honeydew produced by Bemisia babaci feeding on cotton. Plant Physiol. 98 : 753-756
Thinh NT., Takagi H. and Tashima S. ( 1999 ) Cryopreservation of in vitro-grown shoot tips of banana ( Musa spp. ) by vitrification method. Cryo-Letters 20 : 163-174
Towill LE. ( 1990 ) Cryopreservation. In : Dodds JH ( ed. ) In vitro methods for conservation of plant genetic resources. Chapman and Hall, London, pp.41-69
Towill LE. and Jarret RL. ( 1992 ) Cryopreservation of sweet potato [ Ipomoea batatas ( L ) Lam. ] shoot tips by vitrification. Plant Cell Reports 11 : 175-178
Wang HL., Lee PD., Chen WL., Huang DJ. and Su JC. ( 2000 ) Osmotic stress-induced changes of sucrose metabolism in cultured sweet potato cells. Journal of Experimental Botany 51 : 1991-1999
Wang JH. ( 2000 ) A comprehensive evaluation of the effects and mechanisms of antifreeze proteins during low-temperature preservation. Cryobiology 41 : 1-9
Wowk B., Leitl E., Rasch CM., Mesbah-Karimi N., Harris SB. and Fahy GM. ( 2000 ) Vitrification enhancement by synthetic ice blocking agents. Cryobiology 40 : 228-236
Yamada T., Sakai A., Matsumura T. and Higuchi S. ( 1991 ) Cryopreservation of apical meristems of white clover ( Trifolium repens L. ) by vitrification. Plant Science 78 : 81-87
Yoshida S., Forna JH., Cock H., and Gornez KA. ( 1976 ) Determination of chlorophyll in plant tissue. Laboratory manual of physiological studies of rice. IRRI 26 : 43-49