Please use this identifier to cite or link to this item:
標題: 草莓玻璃化法超低溫冷凍保存流程之探討
Investigation of the Protocol on the Cryopreservation of Strawberry (Fragaria x ananassa Duch.) Treated by Vitrification
作者: 黃坤陽
Hwang, Kun-Yang
關鍵字: cryopresevation;超低溫冷凍保存;vitrification;cold acclimation;玻璃化法;冷馴化
出版社: 生命科學系所
引用: 李窓明。1993。 草莓新品種「桃園二號」特性與栽培管理。 桃園區農業專訊(4):7-10。 李窓明。 2003。 草莓本圃栽培管理。 桃園區農業專訊(44):15-21。 李窓明與李聯興。1999。草苺桃園三號之育成。桃園農業改良場研究彙報(39):1- 17。 胡馨分。2006。北蕉玻璃化法超低溫冷凍保存流程之探討。國立中興大學碩士論文。 陳玟君。2006。台農13號鳳梨玻璃化法超低溫冷凍保存前處理之流程探討。國立中興大學生命科學研究所碩士論文。 劉校春。1989。草莓栽培技術。五洲出版社。 劉淑芬。2005。台農十六號鳳梨玻璃化法超低溫冷凍保存前處理流程之探討。國立中興大學生命科學研究所碩士論文。 劉增城。1998。草莓王國-大湖鄉。苗栗區農業專訊。(4):16-19 葛會波。1994。草莓高效益栽培。中國農業出版社。 蔡淑華。1995。植物組織切片技術綱要。茂昌圖書有限公司。pp. 47-58 Ashworth EN, Pearce RS (2002) Extracellular freezing in leaves of freezing-sensitive species. Planta 214: 798-805 Baek HJ, Kim HH, Cho EG, Chae YA, Engelmann F (2003) Importance of explant size and origin and of preconditioning treatments for cryopreservation of garlic shoot apices by vitrification. Cryo-Letters 24: 381-388 Bajaj YPS (1991) Storage and cryopreservation of in vitro cultures. In: Bajaj YPS (ed) Biotechnology in agriculture and forestry, vol 17. High-tech and micropropagation I. Springer, Berlin Heidelberg New York, pp 361-381. Bakht J, Bano A, Dominy P (2006) The role of abscisic acid and low temperature in chickpea (Cicer arietinum) cold tolerance. II. Effects on plasma membrane structure and function. J Exp Bot 57: 3707-3715 Benson EE (1999) Conservation, In: BensonEE (ed), Plant Consevation Biotechnology, Taylor & Francis, London, 1999, pp 83-95. Bhowmik PK, Tamura K, Sanada Y, Tase K, Yamada T (2006) Sucrose metabolism of perennial ryegrass in relation to cold acclimation. Z Naturforsch [C]. 61: 99-104 Bohn M, Luthje S, Sperling P, Heinz E, Dorffling K (2007) Plasma membrane lipid alteration induced by cold acclimation and abscisic acid treatment of winter wheat seedlings differing in frost resistance. J Exp Bot 164: 146-156 Bourion V, Lejeune-Henaut I, Munier-Jolain N, Salon C (2003) Cold acclimation of winter and spring peas: carbon partitioning as affect by light intensity. Europ J Agron 19: 535-548 Chang YJ, Reed BM (1999) Extended cold acclimation and recovery medium alteration improve regrowth of Rubus shoot tips following cryopreservation. Cryo-Letters 20: 371-376 Chang YJ, Reed BM (2000) Extended alternating-temperature cold acclimation and culture duration improve pear shoot cryopreservation. Cryobiology 40: 311-322 Cheel J, Theoduloz C, Rodriguez JA, Caligari PDS, Schmeda-Hirschmann G (2007) Free radical scavenging activity and phenolic content in achenes and thalamus from Fragaria Chiloensis ssp. chiloensis, F. vesca and F. x ananassa cv. Chandler. Food Chem 102: 36-44 Engelmann F (2004) Plant cryopreservation: Progress and Prospects. In Vitro Cell Dev Biol-Plant 40: 427-433 Guy CL, Huber JLA, Huber SC (1992) Sucrose phosphate synthase and sucrose accumulation at low temperature. . Plant Physiol 100: 502-508 Hannum SM (2004) Potential impact of strawberries on human health: a review of the science. Crit Rev Food Sci Nutr. 44: 1-17 Hirai D, Sakai A (2003) Simplified cryopreservation of sweet potato [Ipomoea batatas(L.) Lam.] by optimizing conditions for osmoprotection. Plant Cell Rep 21: 961-966 Hirai D, Shirai K, Shirai S, Sakai A (1998) Cryopreservation of in vitro-grown meristems of strawberry(Fragaria x ananassa Duch.)by encapsulation-vitrification. Euphytica 101: 109-115 Hitmi A, Barthomeuf C, Sallanon H (1999a) Cryopreservation of chrysanthemum cinerariaefolium shoot tips. Effect of pretreatment conditions and retentoin of biosynthetic capacity. Cryo-Letters 20: 109-120 Hitmi A, Coudret A, Barthomeuf C, Sallanon H (1999b) The role of sucrose in freezing tolerance in Chrysanthemum cinerariaefolium L.cell cultures. Cryo-Letters 20: 45-54 Hoekstra FA, Golovina EA, Buitink J (2001) Mechanism of plant desiccation tolerance. Trends Plant Sci 6: 431-438 Houssa P, Bernier G, Kinet JM (1991) Qualitative and quantitative analysis of carbohydrates in leaf exudes of the short-day plant, Xanthium strumarium L. during floral transition. J Plant Physiol 138: 24-28 Jensen WA (1973) Botanical histochemistry W.H.Freeman and Company, San Francisco London pp.55-99 Jitsuyama Y, Suzuki T, Harada T, Fujikawa S (2002) Sucrose incubation increases freezing tolerance of asparagus (Asparagus officinalis L.). Cryo-Letters 23: 103-112 Keller ERJ, Senula A, Leunufna S, Grube M (2006) Slow growth storage and cryopreservation - tools to facilitate germplasm maintenance of vegetatively propagated crops in living plant collections. .Intl J of Refrig 29: 411-417 Lambardi M, Fabbri A, Caccavale A (2000) Cryopreservation of white poplar (Populus alba L.) by vitrification of in vitro-grown shoot tips. Plant Cell Rep 19: 213-218 Leunufna S, Keller ERJ (2005) Cryopreservation of yams using vitrification modified by including droplet method: Effects of cold acclimation and sucrose. Cryo-Letters 26: 93-102 Liu YG, Wang XY, Liu LX (2004) Analysis of genetic variation in surviving apple shoots following cryopreservation by vitrification. Plant Sci 166: 677-685 Livingston DP, Premakumar R, Tallury SP (2006) Carbohydrate partitioning between upper and lower regions of the crown in oat and rye during cold acclimation and freezing. Cryobiology 52: 200-208 Matsumoto T, Sakai A, 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 Na HY, Kondo K (1996) Cryopreservation of tissue-culture shoot primordia from shoot apices od cultured protocorms in Vanda pumila following ABA preculture and desication. Plant Sci 118: 195-201 Niino T, Tanaka D, Ichikawa S, Takano J, Ivette S, Shirata K, Uemura M (2003) Cryopreservation of in vitro-grown apical shoot tips of strawberry by vitrification. Plant Biotechnol 20: 75-80 Paul H, Daigny G, Sangwan-Noreel BS (2000) cryopreservation of apple (Malus x domestica Borkh.) shoot tips following encapsulation-dehydration or encapsulation-vitrification. Plant Cell Rep 19: 768-774 Pearce RS (2004) Adaptation of higher plants to freezing.In Fuller BJ, Lane N, Benson EE (Eds.). Life in the frozen state, CRC Press LLC: 171-203. Rajashekar CB, Zhou H, Marcum KB, Prakash O (1999) Glycine betaine accumulation and induction of cold tolerance in strawberry plants. Plant Sci 148: 175-183 Reed BM, Hummer KE (1995) Conservation of germplasm of strawberry(Fragaria species). In: Bajaj YPS (ed) Biotechnology in agriculture and forestry, vol 32. Cryopreservation of Plant Germplasm I. Springer-Verlag, Berlin Heidelberg New York, pp 354-370 Rosa M, Hilal M, Gonzalez JA, Prado FE (2004) Changes in soluble carbohydrates and related enzymes induced by low temperature during early developmental stages of quinoa (Chenopodium quinoa) seedlings. J Plant Physiol 161: 683-689 Ryynänen L (1998) Effect of abscisic acid, cold hardening, and photoperiod on recovery of cryopreservation in vitro shoot tips of silver birch. Cryobiology 36: 32-39 Ryynänen L, Aronen T (2005) Vitrification, a complementary cryopreservation method for Betula pendula Roth. Cryobiology 51: 208-219 Sakai A (2004) Plant cryopreservation, In B.J. Fuller, N. Lane, E.E. Benson(Eds.). Life in the frozen state, CRC Press LLC: 329-346. Sakai A, Kobayashi S, Oiyama I (1990) Cryopreservation of nucellar cells of navel orange (Citrus sinensis Osb.var. brasiliensis Tanaka) by vitrification. Plant Cell Rep 9: 30-33 Sakai A, Yamakawa M, Sakata D, Harada T, Yakuwa T (1978) Development of whole plant from an excised strawberry runner apex frozen to -196 ℃. Low Temp Sci Ser B 36: 31-38 Sasaki H, Ichimura K, Okada K, Oda M (1998) Freezing tolerance and soluble sugar contents affected by water stress during cold-acclimation and de-acclimation in cabbage seedling. Sci. Hortic. 76: 161-169 Scottez C, Chevreau E, Godard N, Arnaud Y, Duron M, Dereuddre J (1992) Cryopreservation of cold-acclimated shoot tips of pear in vitro cultures after encapsulation-dehydration. Cryobiology 29: 691-700 Slavik B (1974) Water in cells and tissues. In Slavik B(ed) Methods of Studying plant water Relations. Academid Publishing House of the Czevhoslovak Academy of Science Prague, New York .pp1-20. Takagi H, Thinh N, Islam O, Senboku T (1997) Cryopreservation of in vitro-grown shoot tips of taro (Colocasia esculenta (L.) Schoot) by vitrification. 1. Investigation of basic conditions of the vitrification procedue. Plant Cell Rep 16: 594-599 Tarczynski MC, Byrne DN, 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 Thomashow MF (1998) Role of cold-resposive genes in plant freezing tolerance. Plant Physiol 118: 1-7 Thomashow MF (1999) Plant cold acclimation: freezing tolerance genes and regulatory mechanism. Annu Rev Plant Physiol Plant Mol Biol 50: 571-599 Turner SR, Senaratna T, Touchell DH, Bunn E, Dixon KW, Tan B (2001a) Stereochemical arrangement of hydroxyl groups in sugar and polyalcohol molecules as an important factor in effective cryopreservation. Plant Sci 160: 489-497 Turner SR, Senaratna T, Bunn E, Tan B, Dixon KW, Touchell DH (2001b) Cryopreservation of Shoot Tips from Six Endangered Australian Species using a Modified Vitrification Protocol. . Ann Bot 87: 371-378 Uemura M, Steponkus PL (1999) Cold acclimation in plants: Relationship between the lipid composition and the cryostability of the plasma membrane. J Plant Res 112: 245-254 Volk GM, Harris JL, Rotindo KE (2006) Survival of mint shoot tips after exposure to cryoprotectant solution components. Cryobiology 52: 305-308 Volk GM, Walters C (2006) Plant vitrification solution 2 lowers water content and alters freezing behavior in shoot tips during cryoprotection. Cryobiology 52: 48-61 Wang QC, Laamanen J, Uosukainen M, Valkonen JPT (2005) Cryopreservation of in vitro-grown shoot tips of raspberry (Rubus idaeus L.) by encapsulation-vitrification and encapsulation-dehydration. Plant Cell Rept 24: 280-288 Yoshida S, Forno D, Cock J, Gomez K (1976) Determination of sugar and starch in plant tissue. In: Yoshida S (ed), Laboratory Manual for Physiological Studies of Rice. The International Rice Research Institute, Los Baños, The Philippines, pp 46–49.
草莓(Fragaria x ananassa Duch.)為溫帶果樹,適合以低溫冷馴化預培養後進行超低溫冷凍保存。本研究期以桃園一號與桃園三號兩種品系草莓經10℃低溫冷馴化7-60天後,進行玻璃化法超低溫冷凍保存能提升存活率。結果顯示,兩種草莓經過冷馴化30天預培養能明顯提升存活率。桃園一號經冷馴化預培養後以LS處理20分鐘、PVS2處理150分鐘,獲最佳存活率約97%;桃園三號是以LS處理20分鐘、PVS2處理240-270分鐘,存活率可達96%;桃園三號預培養於0.2 M培養基30天後,再以LS處理60分鐘、PVS2處理240分鐘,存活率達87%;兩種草莓未經預培養之對照組存活率最高僅為80%。草莓在預培養期間,頂芽細胞之相對含水量、水分潛勢與滲透潛勢亦下降,顯示有輕微脫水現象;可溶性糖類總量增加,果糖、葡萄糖與蔗糖含量產生變化,其中蔗糖大量累積,以上生理變化皆可提升頂芽細胞之抗凍性,進而反應在超低溫冷凍保存存活率上。因此細胞之水分生理變化與滲透調節作用,可使草莓頂芽適應超低溫冷凍保存前處理,進而進入液態氮保存。而回溫生長後的草莓頂芽組織,外觀與一般培養無異,表示冷凍保存處理並未造成組織變異。綜合以上所述,此研究建立之處理流程極適合桃園一號與桃園三號草莓進行之玻璃化法超低溫冷凍保存。

Strawberry (Fragaria x ananassa Duch.) is one kind of temperate plants; therefore, it could be cryopreserved after preculture with cold acclimation. In this study, it is expected that the survival rates of two cultivars of strawberry cv. ''TY-1'' and ''TY-3'', could be maximized through the process of cold acclimation for 7 to 60 days and vitrificated cryopreservation. The results shows that the survival rates of these two kinds of strawberry were obviously promoted after cold acclimation for 30 days and cryopreservation. At first, the survival rate of these two cultivars without preculture was only 80%. However, the highest sur-vival rate of ''TY-1'' became 97% after the shoot tips were cryopreserved following loaded with loading solution (LS) (20 minutes), dehydrated with plant vitrification solution 2 (PVS2) (150 minutes). As for TY-3, the highest survival rate of TY-3 shoot tips was 96% after the procedure of LS (20 minutes), PVS2 (240-270 minutes) and cryopreservation. In addition, after a 30-day preculture on MS medium with 0.2 M sucrose, the TY-3 shoot tips were loaded for 60 minutes and then dehydrated for 240 minutes. After these treatments and cryopreservation, the survival rate of TY-3 shoot tips was 87%.
Moreover, during the process of preculture, there had been some decrease in the relative water content, water potential, and osmotic potential of the shoot tips, but there was an increase in the amount of soluble sugar content within apex tissue. All of the content of simple sugar, fructose, glucose and sucrose changed. Among which, sucrose was accumulated in shoot tips.As a result, the water status, osmore-gulation and enhance the strawberry shoot tips to adapt to the pretreatment of cryopreservation protocols and then being directly plugged into LN for cryopreservation. The morphology of the rewarmed shoot tips are the same with the normal ones . This outcome means that the regrowth of cryopreserved strawberry shoot tips were not variable after the protocols.
In conclusion, the protocol established in this study could be adapted to the cryopreservaion of two cultivars strawberry TY-1 and TY-3 by vitrifyication.
其他識別: U0005-1807200720493900
Appears in Collections:生命科學系所

Show full item record

Google ScholarTM


Items in DSpace are protected by copyright, with all rights reserved, unless otherwise indicated.