扶芳藤茎尖玻璃化超低温保存及其效果评价
|
摘要
玻璃化法超低温保存生物材料是一门新兴的生物技术,已成为目前最为理想的植物种质资源保存方法。目前,有关植物种质资源玻璃化法超低温保存的研究主要集中在玻璃化超低温保存技术程序的优化;检验再生苗的基因稳定性;利用该技术对植物材料进行脱毒;对超低温保存过程中细胞所发生的一系列超微结构变化进行电镜观察等,尚未见对玻璃化超低温保存再生苗后期的生长适应性进行研究的报道。
本研究以'圆瓣'扶芳藤为实验材料,在建立其无菌繁殖体系的基础上,对组培苗的茎尖进行了玻璃化超低温保存研究,并应用AFLP技术分析了液氮保存苗的基因稳定性。通过对超低温保存再生苗进行露地栽培试验,研究了当年春季移栽再生苗的夏季生长适应性、抗寒以及水分适应性。通过试验得出以下结论:
1.建立了'圆瓣'扶芳藤的玻璃化法超低温保存体系。生长健壮的扶芳藤组培苗继代培养30d后,切取2-3mm的茎尖,于20℃下在2M甘油+0.4M乙二醇混和液中处理20min,然后在0℃下用PVS2玻璃化溶液处理50min,换新鲜PVS2溶液,快速浸入液氮,40℃水浴中解冻70s,用MS+1.2mol/L蔗糖液体培养基洗涤2次,每次1 0min。最后,转入MS+6-BA2.0mg/L的培养基上进行恢复培养,成活率达75%。再生植株与对照相同,可进行正常的继代增殖以及生根以及移栽。
2.研究了'圆瓣'扶芳藤再生苗的夏季生长适应性。对在夏季高温环境中再生苗的生长情况、生理生化指标以及光合、荧光特性进行了测定,结果表明,再生苗的生长状况良好,除株高外,分枝数、分枝长以及叶片数均与对照苗没有显著差异;在越夏过程中,再生苗叶片的电导率、光合色素以及脯氨酸含量的变化与对照无显著差异;再生苗的光合特性(光饱和点、补偿点以及CO2的补偿点、饱和点)与对照没有显著差异,但在测定Pn日变化时,发现“午休”期间再生苗的Pn值7.32μmol/m2·s明显低于对照的9.05μmol/m2·s;再生苗的荧光参数(F0、Fm、Fv、Fv/F0、Fv/Fm)以及ΦPSⅡ的光强响应测定值与对照没有显著差异,Fv/Fm日变化测定值中,13:00的再生苗测定值0.775明显低于对照的0.789。
3.研究了'圆瓣'扶芳藤再生苗的越冬适应性。对当年移栽的'圆瓣'扶芳藤玻璃化超低温保存再生苗在越冬期间的生理指标以及叶绿素荧光特性进行测定。结果表明,再生苗叶片的电导率、丙二醛、超氧阴离子释放速率、SOD酶活性以及渗透条件物质脯氨酸、可溶性糖和可溶性蛋白在越冬期间的变化趋势与对照苗相同。但再生苗与对照的差异表现在:12月-2月份再生苗丙二醛的含量和超氧阴离子的释放速率高于对照苗,SOD酶活性在1月份高于对照,2月份低于对照,而脯氨酸含量在1月份为949.3ug/g,此时明显高于对照775.6ug/g。关于再生苗的荧光特性,研究表明再生苗Fv/Fm和ΦPSⅡ的变化趋势与对照相同,而ΦPSII在1月和2月份明显高于对照。通过电导率测定、拟合方程求解以及水插成活率三项指标来综合评价再生苗的抗冻性,结果表明与对照苗差异不显著。
4.研究了'圆瓣'扶芳藤再生苗土壤水分供应适应性。测定了不同土壤含水量情况下的生长情况和生理指标,包括株高、叶片水分状况、电导率、MDA含量、脯氨酸含量、色素含量变化以及光合、荧光特性等。研究表明,在土壤断水过程中,'圆瓣'扶芳藤再生苗生理变化情况与对照苗相同,但脯氨酸含量在断水后的第20d,速积累到473.84ug/g是第15d的12.3倍,但此时含量要明显低于对照苗的535.19ug/g;再生苗的荧光动力学参数Fv/Fm在土壤水分变化过程中保持不变;而净光合速率随着土壤含水量的适宜程度而变化,株高生长速率也随着土壤干旱程度的加重而变缓,这些指标的变化趋势与对照相同,没有显著差异。
5.采用AFLP技术对'圆瓣'扶芳藤再生苗基因稳定性进行了检验。本试验应用筛选出的10对不同引物组合,对随机选取的15株'圆瓣'扶芳藤玻璃化超低温保存再生苗进行了AFLP分析,结果表明再生苗的AFLP图谱中的条带与对照苗没有差异,对超低温保存1、10、30、60和180d的茎尖再生苗也进行了分析,结果表明保存时间的长短不会影响玻璃化超低温保存茎尖的稳定性。从分子水平验证了玻璃化超低温保存技术的安全性、稳定性以及长期性。
本论文的创新性就在于首次对超低温保存种质进行露地栽培试验,并对其生长适应性进行了系统的研究,在国内首次应用AFLP技术对超低温保存苗的基因稳定性进行了系统而全面的研究,首次实现了'圆瓣'扶芳藤的玻璃化超低温保存。
Vitrification as a new biological technology has been thought the most ideal preservation technique of plant culture and their genetic integrity. Researches about this technology mostly focus on the procedure optimization according to different plant species, few analysis of genetic variation of plant following cryopreservation by vitrification, elimination of viruses from plant, examination the ultrastructural changes during vitrification and so on. To our knowedgae, however, no paper reports about the adaptability of the regeneration plant following their transplantation in the field.
This paper studied the vitrification procedure of E. fortunei 'Orbiculatus' with in vitro grown shoot tips, analyzed the genetic variation of the regeneration plantlets, investigated the adaptation of regeneration plantlets to high temperature of summer, cold of winter and different soil water content after their transplantation in the field. The main results were summarized as follows:
1. Shoot tips excised from healthy in vitro plants of E. fortunei. cv. 'Orbiculatus'were successfully cryopreserved by vitrification. A suitable procedure was established as follows: 30 days after subculture, dissected shoot tips of 2-3mm were loaded in a mixture of 2 mol/L glycerol plus 0.4 mol/L sucrose for 20min at 20 °C. Following that, a highly concentrated cryoprotective solution(PVS2)was then added at 0°C. After dehydration at 0°C for 50min, the shoot-tips were directly plunged into liquid nitrogen. After rapid thawing in a water bath at 40°C for 70s, the tips were washed twice with 1.8ml of 1.2 mol/L sucrose solution for 10min each time and then transferred onto MS medium supplemented with 2.0 mg/L 6-BA for recovery growth. The highest survival of shoot tips was over 75 % .The plantlets could normally proliferate, root and transplantation as the control.
2. Studied the adaptive capacity to summer high temperature of the regeneration plantlets of E. fortunei cv. 'Orbiculatus' .We tested the vegetative growth, physiological parameters, photosynthesis and chlorophyll fluorescence characteristics at intervals. The result showed that no significant differences (p<0.05) were observed in vegetative growth including branch number, total branch length and leaf number. But data of the plant height revealed that the plant height of the contrast was significant higher than the regeneration plantlets and the reason of that was not clear and more researches were needed. As to the physiological parameters (photosynthetic pigment content, proline and electrolyte), no statistical difference were fond between the regeneration plantlets and the contrast. The test of regeneration plantlets about the light compensation point, light saturation point of photosynthesis and the CO_2 compensation point, CO_2 saturation point showed no different with the control plantlets. During the measure of daily patterns of Pn, the average Pn of regeneration plantlets in 12:00～14:00 is 7.32μmol/m2· s statistical lower than the control plantlets 9.05μmol/m2·s. While the chlorophyll fluorescence characteristics(F_0, Fm,Fv, Fv/F_0, Fv/Fm, Φ PS II) of regeneration plantlets are also no different with the control plantlets, only the Fv/Fm in the 13:00 of the regeneration plantlets is much lower than the control plantlets.
3. Determination and evaluation of cold resistance of the regeneration plantlets of E. fortunei ' Orbiculatus' . Studied the physiological mechanism of freezing-resistance and chlorophyll fluorescence characteristics during winter with the one year old regeneration plantlets. The results showed that, the change trends of the physiological parameters including the relative electric conductivity, MDA content, superoxide anion radical, SOD activity, free proline, soluble protein and soluble sugar content of the regeneration plantlets during winter were the same with the control plantlets. The different between the regeneration plantlets and the control plantlets in physiological change during winter are as follows: MDA content and superoxide anion radical release rate of the regeneration plantlets during Dec, Jan. and Feb. are higher than the control, SOD activity of Jan. is higher than the control, while lower than the control in Feb., the content of free proline in the leaves of regeneration plantlets is 949.3ug/g, which is higher than 775.6ug/g of the control. As to the chlorophyll fluorescence characteristics, the trends of Fv/Fm and ΦPSII of regeneration plantlets are the same with the control, while theΦPSII in Jan. and Feb. is higher than the control. The methods of electric conductivity, Logistic equation and growing recovery were used to study the freezing tolerance of regeneration plantlets, the result revealed that there is on different with the control ones.
4. Studied the adaptive capacity of the regeneration plantlets of E. fortunei ' Orbiculatus' under different soil moisture. Physiological indices including water state of leaves, relative permeability of membrane, contents of MDA, free proline, and chlorophyll were measured under different soil water content. The result showed that the physiological changes to drought resistance capacity of regeneration plantlets are the same with the control ones. Except the free proline content in the 20th day, the content of the regeneration plantlets is 473.84ug/g dramatically lower than 535.19ug/g of the control. Chlorophyll fluorescence characteristics Fv/Fm were not change during the experiment. While, with the change of soil relative water content, the Pn and the growth increments of the regeneration plantlets were retarded by water stress, which is the same with the control.
5. Genetic fidelity following cryopreservation was evaluated for E. fortunei 'Orbiculatus' . The PCR-based multi-locus DNA fingerprinting technique Amplified Fragment Length Polymorphism (AFLP) analysis of randomly selected 15 cryopreserved-derived and 15 in vitro grown plantlets with 10 AFLP primer combinations produced multi-locus DNA fingerprint with a total of 886 DNA fragments. No qualitative differences were found in the 30 DNA fingerprints. Genetic fidelity was also assessed following 1d, 10d, 30d, 60d, 180d of storage, we found that there no significantly different across storage times. The results proved that cryopreservation using vitrification is a practical method for long—term storage of plant genetic resource at DNA levels.
The innovations of the paper are as follows: First, systemically studied the adaptive capacity of the regeneration plantlets derived from cryopreserved shoot tips by vitrification after transplantation to the field. Second, general evaluated the genetic fidelity of the regeneration plantlets following cryopreservation by vitrification, Finally, shoot tips excised from E. fortunei ' Orbiculatus' were successfully cryopreserved with the vitrification.
本研究以'圆瓣'扶芳藤为实验材料,在建立其无菌繁殖体系的基础上,对组培苗的茎尖进行了玻璃化超低温保存研究,并应用AFLP技术分析了液氮保存苗的基因稳定性。通过对超低温保存再生苗进行露地栽培试验,研究了当年春季移栽再生苗的夏季生长适应性、抗寒以及水分适应性。通过试验得出以下结论:
1.建立了'圆瓣'扶芳藤的玻璃化法超低温保存体系。生长健壮的扶芳藤组培苗继代培养30d后,切取2-3mm的茎尖,于20℃下在2M甘油+0.4M乙二醇混和液中处理20min,然后在0℃下用PVS2玻璃化溶液处理50min,换新鲜PVS2溶液,快速浸入液氮,40℃水浴中解冻70s,用MS+1.2mol/L蔗糖液体培养基洗涤2次,每次1 0min。最后,转入MS+6-BA2.0mg/L的培养基上进行恢复培养,成活率达75%。再生植株与对照相同,可进行正常的继代增殖以及生根以及移栽。
2.研究了'圆瓣'扶芳藤再生苗的夏季生长适应性。对在夏季高温环境中再生苗的生长情况、生理生化指标以及光合、荧光特性进行了测定,结果表明,再生苗的生长状况良好,除株高外,分枝数、分枝长以及叶片数均与对照苗没有显著差异;在越夏过程中,再生苗叶片的电导率、光合色素以及脯氨酸含量的变化与对照无显著差异;再生苗的光合特性(光饱和点、补偿点以及CO2的补偿点、饱和点)与对照没有显著差异,但在测定Pn日变化时,发现“午休”期间再生苗的Pn值7.32μmol/m2·s明显低于对照的9.05μmol/m2·s;再生苗的荧光参数(F0、Fm、Fv、Fv/F0、Fv/Fm)以及ΦPSⅡ的光强响应测定值与对照没有显著差异,Fv/Fm日变化测定值中,13:00的再生苗测定值0.775明显低于对照的0.789。
3.研究了'圆瓣'扶芳藤再生苗的越冬适应性。对当年移栽的'圆瓣'扶芳藤玻璃化超低温保存再生苗在越冬期间的生理指标以及叶绿素荧光特性进行测定。结果表明,再生苗叶片的电导率、丙二醛、超氧阴离子释放速率、SOD酶活性以及渗透条件物质脯氨酸、可溶性糖和可溶性蛋白在越冬期间的变化趋势与对照苗相同。但再生苗与对照的差异表现在:12月-2月份再生苗丙二醛的含量和超氧阴离子的释放速率高于对照苗,SOD酶活性在1月份高于对照,2月份低于对照,而脯氨酸含量在1月份为949.3ug/g,此时明显高于对照775.6ug/g。关于再生苗的荧光特性,研究表明再生苗Fv/Fm和ΦPSⅡ的变化趋势与对照相同,而ΦPSII在1月和2月份明显高于对照。通过电导率测定、拟合方程求解以及水插成活率三项指标来综合评价再生苗的抗冻性,结果表明与对照苗差异不显著。
4.研究了'圆瓣'扶芳藤再生苗土壤水分供应适应性。测定了不同土壤含水量情况下的生长情况和生理指标,包括株高、叶片水分状况、电导率、MDA含量、脯氨酸含量、色素含量变化以及光合、荧光特性等。研究表明,在土壤断水过程中,'圆瓣'扶芳藤再生苗生理变化情况与对照苗相同,但脯氨酸含量在断水后的第20d,速积累到473.84ug/g是第15d的12.3倍,但此时含量要明显低于对照苗的535.19ug/g;再生苗的荧光动力学参数Fv/Fm在土壤水分变化过程中保持不变;而净光合速率随着土壤含水量的适宜程度而变化,株高生长速率也随着土壤干旱程度的加重而变缓,这些指标的变化趋势与对照相同,没有显著差异。
5.采用AFLP技术对'圆瓣'扶芳藤再生苗基因稳定性进行了检验。本试验应用筛选出的10对不同引物组合,对随机选取的15株'圆瓣'扶芳藤玻璃化超低温保存再生苗进行了AFLP分析,结果表明再生苗的AFLP图谱中的条带与对照苗没有差异,对超低温保存1、10、30、60和180d的茎尖再生苗也进行了分析,结果表明保存时间的长短不会影响玻璃化超低温保存茎尖的稳定性。从分子水平验证了玻璃化超低温保存技术的安全性、稳定性以及长期性。
本论文的创新性就在于首次对超低温保存种质进行露地栽培试验,并对其生长适应性进行了系统的研究,在国内首次应用AFLP技术对超低温保存苗的基因稳定性进行了系统而全面的研究,首次实现了'圆瓣'扶芳藤的玻璃化超低温保存。
Vitrification as a new biological technology has been thought the most ideal preservation technique of plant culture and their genetic integrity. Researches about this technology mostly focus on the procedure optimization according to different plant species, few analysis of genetic variation of plant following cryopreservation by vitrification, elimination of viruses from plant, examination the ultrastructural changes during vitrification and so on. To our knowedgae, however, no paper reports about the adaptability of the regeneration plant following their transplantation in the field.
This paper studied the vitrification procedure of E. fortunei 'Orbiculatus' with in vitro grown shoot tips, analyzed the genetic variation of the regeneration plantlets, investigated the adaptation of regeneration plantlets to high temperature of summer, cold of winter and different soil water content after their transplantation in the field. The main results were summarized as follows:
1. Shoot tips excised from healthy in vitro plants of E. fortunei. cv. 'Orbiculatus'were successfully cryopreserved by vitrification. A suitable procedure was established as follows: 30 days after subculture, dissected shoot tips of 2-3mm were loaded in a mixture of 2 mol/L glycerol plus 0.4 mol/L sucrose for 20min at 20 °C. Following that, a highly concentrated cryoprotective solution(PVS2)was then added at 0°C. After dehydration at 0°C for 50min, the shoot-tips were directly plunged into liquid nitrogen. After rapid thawing in a water bath at 40°C for 70s, the tips were washed twice with 1.8ml of 1.2 mol/L sucrose solution for 10min each time and then transferred onto MS medium supplemented with 2.0 mg/L 6-BA for recovery growth. The highest survival of shoot tips was over 75 % .The plantlets could normally proliferate, root and transplantation as the control.
2. Studied the adaptive capacity to summer high temperature of the regeneration plantlets of E. fortunei cv. 'Orbiculatus' .We tested the vegetative growth, physiological parameters, photosynthesis and chlorophyll fluorescence characteristics at intervals. The result showed that no significant differences (p<0.05) were observed in vegetative growth including branch number, total branch length and leaf number. But data of the plant height revealed that the plant height of the contrast was significant higher than the regeneration plantlets and the reason of that was not clear and more researches were needed. As to the physiological parameters (photosynthetic pigment content, proline and electrolyte), no statistical difference were fond between the regeneration plantlets and the contrast. The test of regeneration plantlets about the light compensation point, light saturation point of photosynthesis and the CO_2 compensation point, CO_2 saturation point showed no different with the control plantlets. During the measure of daily patterns of Pn, the average Pn of regeneration plantlets in 12:00～14:00 is 7.32μmol/m2· s statistical lower than the control plantlets 9.05μmol/m2·s. While the chlorophyll fluorescence characteristics(F_0, Fm,Fv, Fv/F_0, Fv/Fm, Φ PS II) of regeneration plantlets are also no different with the control plantlets, only the Fv/Fm in the 13:00 of the regeneration plantlets is much lower than the control plantlets.
3. Determination and evaluation of cold resistance of the regeneration plantlets of E. fortunei ' Orbiculatus' . Studied the physiological mechanism of freezing-resistance and chlorophyll fluorescence characteristics during winter with the one year old regeneration plantlets. The results showed that, the change trends of the physiological parameters including the relative electric conductivity, MDA content, superoxide anion radical, SOD activity, free proline, soluble protein and soluble sugar content of the regeneration plantlets during winter were the same with the control plantlets. The different between the regeneration plantlets and the control plantlets in physiological change during winter are as follows: MDA content and superoxide anion radical release rate of the regeneration plantlets during Dec, Jan. and Feb. are higher than the control, SOD activity of Jan. is higher than the control, while lower than the control in Feb., the content of free proline in the leaves of regeneration plantlets is 949.3ug/g, which is higher than 775.6ug/g of the control. As to the chlorophyll fluorescence characteristics, the trends of Fv/Fm and ΦPSII of regeneration plantlets are the same with the control, while theΦPSII in Jan. and Feb. is higher than the control. The methods of electric conductivity, Logistic equation and growing recovery were used to study the freezing tolerance of regeneration plantlets, the result revealed that there is on different with the control ones.
4. Studied the adaptive capacity of the regeneration plantlets of E. fortunei ' Orbiculatus' under different soil moisture. Physiological indices including water state of leaves, relative permeability of membrane, contents of MDA, free proline, and chlorophyll were measured under different soil water content. The result showed that the physiological changes to drought resistance capacity of regeneration plantlets are the same with the control ones. Except the free proline content in the 20th day, the content of the regeneration plantlets is 473.84ug/g dramatically lower than 535.19ug/g of the control. Chlorophyll fluorescence characteristics Fv/Fm were not change during the experiment. While, with the change of soil relative water content, the Pn and the growth increments of the regeneration plantlets were retarded by water stress, which is the same with the control.
5. Genetic fidelity following cryopreservation was evaluated for E. fortunei 'Orbiculatus' . The PCR-based multi-locus DNA fingerprinting technique Amplified Fragment Length Polymorphism (AFLP) analysis of randomly selected 15 cryopreserved-derived and 15 in vitro grown plantlets with 10 AFLP primer combinations produced multi-locus DNA fingerprint with a total of 886 DNA fragments. No qualitative differences were found in the 30 DNA fingerprints. Genetic fidelity was also assessed following 1d, 10d, 30d, 60d, 180d of storage, we found that there no significantly different across storage times. The results proved that cryopreservation using vitrification is a practical method for long—term storage of plant genetic resource at DNA levels.
The innovations of the paper are as follows: First, systemically studied the adaptive capacity of the regeneration plantlets derived from cryopreserved shoot tips by vitrification after transplantation to the field. Second, general evaluated the genetic fidelity of the regeneration plantlets following cryopreservation by vitrification, Finally, shoot tips excised from E. fortunei ' Orbiculatus' were successfully cryopreserved with the vitrification.
引文
1．Katakawa J．扶芳藤茎叶中的三萜烯．《国外医学：中医中药分册》，2002，24(1)：46
2．艾鹏飞，卢利平．桔梗试管苗苇尖玻璃化法超低温保存及植株再生[J]．中草约，2006，37(9)：1409-1412
3．艾鹏飞，罗正荣．柿休眠芽茎尖玻璃化法超低温保存及植株再生[J]．中国农业科学，2003，36(5)：553-556
4．敖妍，鲁韧强，潘青华．扶芳藤快速扩繁试验[J]．林业科技开发，2006，21(1)：36-39
5．蔡小宁，陈舒泛，陈俊，等．小球藻的玻璃化超低温保存[J]．植物生理学通讯，2004，40(5)：599-601
6．蔡祖国，徐小彪，赵一鹏，等．猕猴桃茎尖超低温保存过程中超微结构观察[J]．西北植物学报，2006，26(8)：1600-1604
7．陈勇，王君晖，黄纯农．铁皮石斛种质资源的玻璃化法超低温保存[J]．浙江大学学报(农业与生命科学版)，2001，27(4)：436-438
8．陈黎．复方扶芳藤合剂抗大肠癌术后化疗白细胞减少疗效观察[J]．广西中医药，2001，24(5)：297-298
9．陈品良．植物组织培养物的超低温保存[J]．武汉植物研究，1989，7(4)：390-398
10．陈书安，王晓东，赵兵，等．水母雪莲愈伤组织超低温保存条件的初探[J]．过程工程学报，2002，2(6)：539-543
11．陈勇，陈娴婷，王君晖．瓯柑愈伤组织的玻璃化法超低温保存研究[J]．浙江大学学报(理学版)，2004，31(2)：197-201
12．陈勇，王君晖，黄纯农．胡萝卜悬浮培养细胞和原生质体的玻璃化法超低温保存[J]．浙江大学学报(理学版)，2002，29(1)：94-98
13．邓家刚．复方扶芳藤合剂异病同治举偶[J]．广西中医药，2000，23(3)：47-48
14．董丽，黄亦工，贾麦娥，等．北京园林主要常绿阔叶植物抗冻性及其测定方法[J]．北京林业大学学报，2002，24(3)：70-73
15．高建钰，马晶，樊明瑞，等．扶芳藤营养袋育苗试验[J]．山西林业科技，2005，1：12-14
16．葛锋，王晓东，赵兵，等．肉从蓉愈伤组织的超低温保藏方法[J]．过程工程学报，2006，6(5)：794-798
17．关雪莲，马清水，张宁，等．大叶黄杨(Euonymus japonicus)和扶芳藤(Euonymus fortunei)耐寒生理指标的变化．北京农学院学报，2006，7：29-33
18．郭燕霞，刘玉军．长鞭红景大悬浮培养细胞的玻璃化法超低温保存研究[J]．西北植物学报，2006，26(8)：1605-1611
19．胡明珏，王君晖．拟南芥悬浮细胞系的玻璃化法超低温保存[J]．细胞生物学杂志，2004，26(1)：81-84．
20．黄纯农，王君晖，颜秋生，等．大麦胚性悬浮细胞系的快速建立及其超低温保存[J]．中国农业科学，1995，28(3)：21-27
21．黄纯农，颜秋生．玻璃化法液氮冻存大麦和水稻的胚性悬浮细胞[J]．杭州大学学报：自然科学版，1994，21(1)：114-115
22．简令成．低温生物学与植物种质的K期保存[J]．植物学通报，1988，5(2)：65-68
23．缴丽莉，路丙社，白志英，等．四种园林树木抗寒性的比较分析[J]．园艺学报，2006，33(3)：667-670
24．金万梅，尹淑萍，鲁韧强，等．扶芳藤组织培养再生体系的建立[J]．植物生理学通讯，2005，41(1)：27-30
25．李广武、郑从义等．低温生物学[M]．长沙：湖南科技出版社，1998
26．李合生．植物生理生化实验原理和技术．北京：高等教育出版社，2000，258-260
27．李吉跃．太行山区主要造林树种耐早特性的研究[D1]．北京林业大学博士学位论文，1990
28．李锦树，王洪春，王文英．1983．干旱对玉米叶片细胞膜透性及膜脂的影响．植物生理学通讯，1983，9：223-228
29．李明军，洪森荣，徐鑫，等．怀山药种质资源的玻璃化法超低温保存[J]．作物学报，2006，2，288-292
30．李鹏，李航．扶芳藤中的三萜化合物研究[J]．中国药学杂志，2000，35(12)：847-848
31．李雪芹，徐礼根，金松恒，马博英．4种草坪草叶绿素荧光特性的比较[J]．园艺学报，2006，33(1)：164-167
32．林田，刘灶长，李天菲，等．红花石蒜茎尖的玻璃化超低温保存[J]．植物生理学通讯，2006，42(6)：1063-1066
33．刘长利，甘草抗旱特性的初步研究，河北农业大学，硕士学位论文，2002
34．刘峰，颜秋生．水稻胚性悬浮细胞玻璃化冻存中的超微结构变化[J]．中国水稻科学，1998，12(1)：17-20
35．刘小攀，姜超．利用槐荫扦插扶芳藤[J]．林业实用技术，2003，5：24
36．刘燕，高荣孚，周晓刚．玻璃化超低温保存中拟南芥幼苗细胞膜ATPase的变化[J]．北京林业大学学报，2001，23(5)：1-5
37．刘燕．拟南芥幼苗玻璃化超低温保存研究．北京林业大学，博士论文，2000
38．刘云国，王晓云．苹果种质资源玻璃化法超低温保存技术[J]．山东农业大学学报(自然科学版)，2002，33(1)：32-36
39．刘云国．苹果种质资源玻璃化法超低温保存技术及其遗传变异分析．山东农业大学，博士论文．
40．罗俊，张木清，林彦铨，张华，陈如凯，甘蔗苗期叶绿素荧光参数与抗旱性关系研究[J]．中国农业科学 2004，37(11)：1718-1721
41．马锋旺，李嘉瑞．杏原生质体的超低温[J]．园艺学报．1998，25(4)：329-332
42．梅俊学．逆温下发菜脯氨酸含量及膜透性的变化与含水量的关系．山东师大学报(自然科学版)，15(2)：178-181
43．孟庆辉，潘青华，鲁韧强，等．4个品种扶芳藤茎叶解剖结构及其与抗旱性的关系[J]．农业基础科学，2006，22(4)：138-142
44．潘青华，宋婉，鲁韧强，等．扶芳藤种质资源及变异研究[J]．北京林业大学学报，2004，26(2)：58-62
45．潘青华．扶芳藤遗传多样性研究及抗逆性选择．北京林业大学，2003，博士论文
46．彭志红，彭克勤，胡家金，萧浪涛．渗透胁迫下植物脯氨酸积累的研究进展[J]．中国农学通报，2002，18(4)：80-83。
47．曲宝军，李大亮．扶芳藤对不同依附体的攀援能力试验[J]．山东林业科技，2001，135(4)：35
48．邵建柱．苹果等果树种质资源的离体保存研究．华中农业大学，博士论文，2003
49．史燕山，李嘉，李长槐，等．生长素对花叶扶芳藤扦插成活及苗质的影响[J]．天津农业科学，2004，10(1)：4-6
50．苏文华，张先辉，王崇云，等．短葶飞蓬光合生理生态的初步研究[J]．云南大学学报，2001，23：142-145
51．唐连顺，李广敏．水分胁迫下玉米叶肉细胞超微结构的变化及其与膜脂过氧化伤害的关系[J]．植物学报，1994，36(增刊)：43-49
52．王和祥，么秀文．木本地被植物引种栽培的研究[J]．园林科技通讯，1997，28：2-10
53．王建 药用植物扶芳藤的组织培养．广西中医学院学报，2004，7(2)：62-63
54．王君晖，严庆丰，黄纯农．大麦幼穗的玻璃化法超低温保存及冻后植株再生[J]．植物学报，1996，38(9)：730-734
55．王君晖，张毅翔，刘峰，等．铁皮石斛种子、原球茎和类原球茎体的超低温保存研究[J]．园艺学报，1999，26(1)：59-61
56．王君晖，郑勇．水稻胚性悬浮细胞的玻璃化超低温保存和可育植株再生[J]．科学通报，1996，41(22)：2081-2084
57．王君辉，黄纯安．玻璃化法-园艺作物茎尖和分生组织超低温保存的新途径[J]．园艺学报，1994，21(3)：277-282．
58．王茂良，赵粱军，任桂芳，等．扶芳藤再生体系的建立[J]．园艺学报，2004，31(2)：241-244
59．王艳军，李锡香，向长萍，等．大蒜茎尖玻璃化法超低温保存技术研究[J]．园艺学报，2005，32(3)：507-509
60．王雁，马武昌．扶芳藤、紫藤等7种藤本植物光能利用特性及耐荫性比较研究[J]．林业科学研究，2004，17(3)：305-309
61．王永林．马铃薯茎尖超低温保存研究[J]．甘肃农业科技，2006(10)：3-5
62．王子成，邓秀新．柑橘体细胞杂种超低温保存后的植株再生．园艺学报，2004，31(2)：215-216
63．王子成，邓秀新．玻璃化法超低温保存柑桔茎尖及植株再生[J]．园艺学报，2001，28(4)：301-306
64．王家福，刘月学．枇杷茎尖二步玻璃化法超低温保存的研究[J]．植物资源与环境学报，2006，15(2)：75-76
65．吴黎明，曾继吾，彭抒昂，等．香蕉茎尖超低温保存过程中的细胞超微结构观察[J]．分子细胞生物学，2006，39(6)：563-567
66．吴庆华，李春霞．扶芳藤直接扦插栽培初探[J]．中国野生植物资源，2005，24(3)：59
67．伍小燕，黄莹娟．扶芳藤对血栓形成及凝血系统的影响[J]．西北药学杂志，1997，12(1)：19-20
68．项黎新，邵健忠．衣藻细胞坡璃化超低温保存技术的研究[J]．细胞生物学杂志，2001，110-113
69．肖洁凝，黄学林．茎尖利芽的超低温保存[J]．生物工程进展，1999，19(5)：46-51
70．谢金鲜，林启云．扶芳藤对心血管作用的试验研究[J]．广西中医药，1999，51-53
71．徐刚标．植物种质资源离体保存研究进展[J]．中南林学院学报，2000，20(4)：81-87
72．薛建平，张爱民，柳俊，等．玻璃化法超低温保存地黄茎尖[J]．农业生物技术学报，2003，11(4)：430-431
73．严庆丰，黄纯农．植物组织和细胞的玻璃化冻存研究[J]．细胞生物学杂志，1994，16(3)：117-122
74．阎秀峰，孙国荣，肖玮．星星草光合蒸腾特性的季节变化[J]．植物研究，1996，16(3)：340-345
75．殷晓辉，舒理慧，郑从义，等．野生稻愈伤组织的超低温保存和冻后再生植株的形成[J]．武汉植物学研究，1996，14(3)：247-252
76．喻方圆，徐锡增．植物逆境生理研究进展．世界林业研究，2003，16(5)：6-11
77．曾继吾，易干军，张秋明，等．番术瓜茎尖超低温保存过程中的细胞超微结构[J]．园艺学报，2005，32(1)：15-19
78．曾继吾，易干军，张秋明，等．果树种质资源离体保存研究进展[J]．果树学报，2002，19(5)：302-306
79．翟发林，丁青龙，张汉民．扶芳藤化学成分研究[J]．南京军医学院学报，2001，23(4)：221-223，226
80．张德舜，刘红权．从种常绿阔叶树种抗寒性的研究[J]．园艺学报，1994，21(3)：283-287
81．张明生，谢波，谈锋，张启堂．甘薯可溶性蛋白、叶绿素及ATP含量变化与品种抗旱性关系的研究[J]．中国农业科学，2003，36(1)：13-16
82．张丕方，倪德祥，包慈华．植物组织培养与繁殖上的应用[M]。上海教育出版社，1985：45-48
83．张守梅，李建国，陈厚彬，等．香蕉茎尖滴冻法保存中细胞的超微结构变化[J]．园艺学报，2006，33(4)：709-713
84．张守仁．叶绿素荧光动力学参数的意义及讨论．植物学通讯，1999，16(4)：444-448
85．张演义，孙仲序，刘建强等．超低温保存果树种质资源研究进展[J]．山东林业科技，2002，138(1)：45-47
86．张玉进，张兴国，庞杰，等．魔芋茎尖玻璃化冻存研究[J]．作物学报，2001，27(1)：97-102
87．张玉进、张兴国，刘佩瑛，等．植物茎尖的玻璃化冻存研究[J]．武汉植物学研究，1999，17(增刊)：78-82
88．张玉芹，李锡香，马庆．实用百合种质的玻璃化法超低温保存技术初探[J]．中国蔬菜，2004(4)：11-13
89．赵黎芳，张金政，张启翔，等．水分胁迫下扶芳藤幼苗保护酶活性和深透调节物质的变化[J]．植物研究，2003，23(4)：437-442
90．赵黎芳，张金政，张启翔，等．盐和水分预处理对扶芳藤幼苗抗寒性的影响[J]．植物研究，2004，24(3)：313-316
91．赵艳华，吴雅琴，李春敏．梨离体茎尖超低温保存方法的比较研究[J]．河北农业科学，2004，8(4)：93-95
92．赵艳华，吴雅琴．桃离体茎尖的超低温保存及植株再生[J]．园艺学报，2006，33(5)：1042-1044
93．赵艳华，吴永杰，周明德．马哈利樱桃离体茎尖超低温保存的研究[J]．园艺学报，1999，26(6)：402-403
94．郑国生，何秀丽．夏季遮荫改善大田牡丹叶片光合功能的研究[J]．林业科学，2006，42(4)：27-32
95．周小梅，王国英，敖光明，等．玉米组培材料的玻璃化法超低温保存及冻后植株再生[J]．农业生物技术学报，2001，9(4)：355-358
96．周雪玲，付超，陈奇凌．扶芳藤组织培养技术研究[J]．新疆农业科学，2006，1：47
97．朱红梅，钟鸣，黄琳芸，等．扶芳藤及其提取物有关药理作用的实验研究[J]．中国中医药科技，2000，7(3)：170
98．朱西存，卢胜西，时鑫．扶芳藤特性及扦插繁育技术[J]．山东林业科技，2004，152(3)：42
99. Ahn Y H, and Sakai A. Survival by vitrification of Sendtanthma grandiforum L. shoot tips for cryopreservation. Horticultural Science, 1994, 35(5): 499-506
100. Angelika SM, Schumacher HM, Gunda MW. Long-term storage of old patato varieties by cryopreservation of shoot-tips in liquid nitrogen. Plant Genetic Resource Newsletter, 1997, (111): 19-24
101. Arnon D I. Copper enzymes in isolated chloroplasts. Polyphenoloxidase in Beta vulagris. Plant Physiology, 1949, 24:1-15
102. Baek H J, Kim H H, Cho E G. et al. Importance of explant size and origin and of preconditioning treatments for cryopreservation of garlic shoot apices by vitrification[J]. Cryo-letters. 2003,24(6):381-388
103. Beadle C L, Ludlow M M, Honeysett J L. In:Hall, E. O., J. M. O. Scurlock & H. R. Bolhar Norderkampf eds. Photosynthesis and production in a changing environment. A field and laboratory manual. 1993, London :Chapman & Hall.
104. Ben G Y, Osmond C B and Sharkey T D. Comparisons of photosynthetic responses of Xanthium strumarium and Helianthus annuus to chronic and acute water stress in sun and shade[J]. Plant Physiology, 1987, 84:476-482.
105. Benelli C, De Carlo A, Lambardi M, et al. Vitrification of shoot tips, nodal segments and embryogentic tissue of olive (Olea europaea L.) for germplasm cryopreservation[J]. Acta horticulturae. 2001,560:137-140
106. Bowler C, Montagu M V, Inze D. Superoxide dismutase and stress tolerance[J]. Annual Review of Plant Physiology and Plant Molecular Biology, 1992,43:83-116.
107. Channuntapipat C, Collins G, Bertezzi T, et al. Cryopreservation of in vitro almond shoot tips by vitrification[J]. Journal of horticultural science & biotechnology. 2000,75(2):228-232
108. Charoensub R, Phansiri S, Yongmanitchai W, et al. Routine cryopreservation of in vitro-grown axillary apices of cassava (Manihot esculenta Crantz) by vitrification: importance of a simple mononodal culture[J]. Scientia Horticulturea.2003,98: 485-492
109. Cornic G J and Briantais J M. Partitioning of photosynthetic CO2 and O2 reduction in a C3 leaf (Phaseolus vulgaris L.)at different CO2 concentrations during drought stress[J]. Planta 1991,183:178-184
110. Escobar R H, Mafia G, Roca W M. A methodology for recovering cassava plants from shoot tips maintained in liquid nitrogen. Plant Cell Reports. 1997,16,474-478
111. Crowe J H, Crowe L M. Carpenter J ￥,et al. Interaction of sugars with membranes. Biochim. Biophys. Acta, 1989 947:367-384
112. Crowe J H,Carpenter J F,Crowe L M,et al. Are freezing and dehydration similar stress vectors? A comparison of modes of interaction of stailizing solutes with biomolecules. Cryobilogy, 1990,27:219-231
113. Crowe L M, Crowe J H. Stabilisation of membranes in anhydrobiotic organisms. In: Leopld A C (ed) Memberanes, metabolism, and dry organisms. Comstock, Cornell University, New York, 1986,210-230
114. DeVerno L L, Park Y S, Bonga J M, et al. Somaclonal variation in cryopreserved embryogenic clones of white spruce. Plant Cell Reports, 1999, 18(11):948-953
115. Engelmann F, Aguilar M E, Dambier D, et al. Advanteges of cryopreservation of cell suspensions and embryogenic calli for citrus breeding proprammers. Fruit, 1994,49(1):23-30
116. Halmagyi & Pinker. Plant regeneration from Rosa shoot tips cryopreserved by a combined droplet vitrification method [J]. Plant cell, Tissue and Organ culture. 2006,84(2): 145-153
117. Halmagyi A, Fisher-Kluver G, Mix-Wagner, et al. Cryopreservation of Chrysanthemum morifolium (Dendranthema grandiflora Ramat.) using different approaches[J]. Plant cell reports.2004,22(6):371 -375
118. Helliot B, Panis B, Poumay Y, et al. Cryopreservation for the elimination of cucumber mosaic and banana streak viruses from banana (Mus aspp.) Plant Cell Reports. 2002,20:1117-1122
119. Helliot B, Swennen R, Poumay Y, et al. Ultrastructural changes associated with cryopreservation of banana (Musa spp.) highly proliferating menstems. Plant Cell Reports, 2003,21 (7): 690-698
120. Hely M. Haggman, Leena A. Ryynanen, Tuija S. et al. Cryopreservation of embryogenic cultures of Scots pine[J]. Plant cell, tissue and organ culture.1998(54):45-53
121. Hirai D and Sakai A.Cryopreservation of in Vitro-grown meristems of patotato(Solanum tuberosum L.) by encapsulation-vitrification[J].Patato Research. 1999,42:153-160
122. Hirano T, Godo T, Mii M. et al. Cryopreservation of immature seeds of Bletilla striata by vitrification[J]. Plant cell reports. 2005,23(8):534-539
123. Hubscher S L, Touchell D H,C-J Tsai. Cryopreservation of transgenic populus tremuloides using a modified vitrification protocol. In vitro Cellular & Developmental Biology; Spring 2004; 40, ProQuest Biology Journals, pg. 73
124. Ishikawa K, Harata K, Mii M, et al. Cryopreservation of zygotic embryos of a Japanese terrestrial orchid (Bletilla striata) bsy vitrification[J]. Plant Cell Reports, 1997, 16 (11): 754-757
125. Ishikawa K,Harata K,Sakai A, et al. Cryopreservation of zygotic embryos of a Japanese terrestrial orchid (Blettilla striata) by vitrification[J], Plant Cell Reports. 1997,16(11):754-757
126. Ishikawa M, Tandon P, Suzuki M, et al. Cryopreservation of bromegrass (Bromus inermis Leyss) suspension cultured cells using slow prefreezing and vitrification procedures[J]. Plant Science, 1996, 120: 81-88
127. Ishikawa M,Suzuki M,Kishimoto T, et al. Effect of growth phase on survival of bromegrass suspension cells following cryopreservation and abiotic stresses[J] .Annals of botany. 2006, 97(3):453-459
128. Jung D W, Sung C K, Touno K, et al. Cryopreservation of Hyoscyamus niger adventitious roots by vitrification. J. Plant Physiology, 2001, 158: 801-805
129. Kanchit T. Cryopreservation of seeds of Thai orchid (Doritis pulcherrima Lindl.) by vitrification[J]. Cryo letter.2000,21:237-244
130. Kohmura H, Ikeda Y, Sakai A. Cryopreservation of apical meristems of Japanese shallot (Allium wakegi A.) by vitrification and subsequent high plant regeneration[J]. Cryo-letters. 1994,15(5):289-298
131. Kohmura H, Saka i A, Chokyu S, et al. Cryopreservation of in vitro-cultured multiple bud clusters of asparagus (Asparagus officinalis L. cv. Hiroshimagreen (2n=30)) by the techniques of vitrification[J]. Plant Cell Reports, 1992, 11: 433-437
132. KuranukiY, Sakai A, Cryopreservation of intro-grown shoot tips of tea (Camellia sinensis)by vitrification[J]. Cryo-letters. 1995,16(6):345-351
133. Lambardi M, Fabbri A, Caccavale A.Cryopreservation of white poplar (Populus alba L.) by vitrification of in vitrogrown shoot tips[J]. Plant Cell Reports.2000, 19:213-218
134. Langis R, Schnabel-Preikstas B, Earle ED, Steponkus P L. Cryopreservation of Brassica compestris cell suspensions by vitrification[J]. Cryo-letters, 1989, 10: 421-428
135. Langis R, Stepinkus P L. Vitrification of ioslated rye protoplasts: protection against dehydration injury by ethylene glycol[J].Cryo-letters.l991,12(2):107-112
136. Langis R, Steponkus P L. Cryopreservation of rye protoplasts by vitrification[J]. Plant Physiology[J].1990, 92, 666-671
137. Leunufna S, Keller E R J. Investigating a new cryopreservation protocol for yams (Dioscorea spp.) [J].Plant Cell Reports.2003,21:1159-1166
138. Lichtenthaler H K . Application of chlorophyll fluorescence in research stress physiology [M],hydrobiology and remote sensing Kluwer Academic Punlishers,Dordrecht, 1991,253-258
139. Liu H Yu, W Dai, J Gong, et al. Cryopreservation of protoplasts of the alga Porphyra yezoensis by vitrification[J]. Plant science. 2004,166(1): 97-102
140. Liu Y G, Wang X Y, Liu L X. Analysis of genetic variation in surviving apple shoots following cryopreservation by vitrification[J]. Plant science. 2004 Mar,166(3):677-685
141. Luo J, Reed B M. Abscisic acid-responsive protein, bovine serum albumin, and praline pretreatments improve recovery of in vitro currant shoot-tip meristems and callus cryopreserved by vitrification[J] .Cryopreservation. 1997, 34:240-250
142. Luyet,B.J. Biodymanica.l937,1:1-14
143. MacFarlane D R,Devitrification in grass forming aqueous solutions,Cryobiology,1986,23: 230-244
144. Maruyama E, Kinoshita I,Ishii K,Ohaba K. and Sakai A. Gemplasm convervation of Guazuma crinita,a useful tree in peru-Amazon,by the cryopreservation of in vitro-cultured multiple bud clusters[J].Plant Cell Tissue and Organ Culture.l997,43(3):161-163
145. Matsumoto T, Mochida K, Itamura H, et al. Cryopreservation of persimmon (Diospyros kaki Thunb.)by vitrification of dormant shoot tips[J].Plant Cell Reports.2001,20:398-402
146. Matsumoto T, Nako Y, Takahashi C,et al. Induction of in vitro cultured masses of shoot primordia of hybrida satatice and its cryopreservation by vitrification[J]. HortScience. 1997,32(2):309-311
147. Matsumoto T, Sakai A, Yamada K Cryopreservation of in vitro-grown apical meristems of lily by vitrification. Plant Cell Tissue Organ Culture., 1995, 41: 237-241
148. Matsumoto T, Sakai A, Yamada K. Cryopreservation of in vitro-grown apical meristems of wasabi (Wasabia japonica) by vitrification and subsequent high plant regeneration[J]. Plant Cell Reports. 1994,13:442-446
149. Matsumoto T, Sakai A. Cryopreservation of axillary shoot tips of in vitro-grown grape(Vitis) by a two-step vitrification protocol[J]. Euphytica: international journal of plant breeding.2003,131 (3):299-304
150. Matsumoto T, Takahashi C, Sakai A, et al. Cryopreservation of in vitro-grown apical meristems of hybrid statice by three different procedures. Scientia Horticture., 1998, 76: 105-114
151. Na H Y, Kondo K. Cryopreservation of tissue-cultured shoot primordia from shoot apices of cultured protocorms in Vanda pumila following ABA preculture and desiccation[J] .Plant Science. 1996,118:195-201
152. Niino T, Sakai A, Yakuwa H, et al. Cryopreservation of in vitro-grown shoot tips of apple and pear by vitrification.Plant Cell[J] .Tissue and Organ Culture. 1992,28:261-266
153. Niino T,Tashiro K, Suzuki M. et al. Cryopreservation of in vitro grown shoot tips of herry by one-step vitrification[J]. cientia horticulturae.l997,July,70(2-3):155-163
154. Nishizawa S, Sakai A, AmanoY. Cryopreservation of Asparagus (Asparagus officinalis L.) embryogenic suspension cell and subsequent plant regeneration by vitrification. Plant Science 1993,91:67-73
155. Panis B, Piette B, Swennen R. Droplet vitrification of apical meristems: a cryopreservation protocol applicable to all Musaceae[J].Plant science. 2005,168(1):45-55
156. Panis B, Totté N, Nimmen K V, et al. Cryopreservation of banana (Musa spp.) meristem cultures after preculture on sucrose[J]. Plant Science. 1996,121:95-106
157. Pennycooke JC, Towill LE. Cryopreservation of shoot tips from in vitro plants of sweet potato (Ipomoea batatas L.) by vitrification[J]. Plant Cell Reports.2000, 19:733-737
158. Polge C. and Smith A.U. Reviaval of spermatozoa after vitrifiation and dehydration at low temperature[J]. Nature. 1949,164:666
159. Rall W F and Fahy G M,Nature.l985,313:573-575
160. Reed B M and Yu X. Cryopreservation of in vitro-grown gooseberry and currant meristems. CryoLetters. 1995,16: 131-136
161.Reed BM, Dumet D, Denoma JM. Validation of cryopreservation protocols for plant germplasm conservation: a pilot study using Ribes L [J]. Biodiversity and Conservation, 2001, 10: 939-949
162. Reinhoud P J, Schrijinemarkers E W M, Iren F.et al. Vitrification and heat-shock treatment improve cryopreservation of tobacco cell suspensions compared to two-step freezing[J]. Plant cell, tissue and organ culture.1995,42(3):261-267
163. Sakai A, Kobayashi S, Oiyama I. Cryopreservation of nucellar cells of navel orange (Citrus sinensis var. brasiliensis Tanaka) by vitrification. Plant Cell Reports, 1990, 9: 30-33
164. Sakai A, Niino T, Yamada T, Kohmura H. Cryopreservation by vitrification of mulberry meristematic callus of white clever and multiple bud clusters of asparagus. Cryobiology, 1992, 29 (6): 747-748
165. Ahuja S, Mandal B B, Dixit S, et al. Molecular, phenotypic and biosynthetic stability in Dioscorea floribunda plants derived from cryopreserved shoot tips[J] .Plant Science.2002,163:971-977
166. Sarkar D,Naik P S. Cryopreservation of shoot tips of tetraploid potato (Solarium tuberosum L.) clones by vitrification[J]. Annals of Botany. 1998,82:455-461
167. Schafer-Menuhar A,Schumarcher H and Wangner G. Long—term storage of old potato varieties by cryopreservation of shoot-tips in liquid nitrogen[J]. Plant Gen. Res. News Lett. 1997,111:19-24
168. Scheuermann,R.,K,Bichler,T. Stuhlfauth and H. Fork. Simultaneous gas exchange and fluorescence measurements indicate differences in the response of sunflower,bean and maize to water stress[J].Photosynthesis Research. 1991,27:189-197
169. Sellferheld M J, Stushnoff C, Fitzpatrick J, et al. Cryopreservation of dormant buds after cold-tender taxa using a modified vitrification procedurer, Hortscicence,1992,27(12):1262
170. Skirvin R M, McPheeters K,Norton M. Sources and frequency of somaclonal variation[J]. Horscience. 1994,29:1232-1235
171. Steponkus P L, Langis R, Fujikawa S. Cryoprservation of plant tissue by vitrification. In: Advances in Low-temperature Biology, edited by Steponkus P.L. JAI press, Ltd., London, 1992, 1: 1-6
172. Tsukazaki H, Mii M, Tokuhara K, et al. Cryopreservation of Doritaenopsis suspension culture by vitrification[J]. Plant cell report.2000, 19(12):1160-1164
173. Takagi H, Thinh NT, Islam OM, et al. Cryopreservation of in vitro-grown shoot tips of taro (Colocasia esculenta (L.) Schott) by vitrification. 1.Investigation of basic conditions of vitrification procedure[J].Plant Cell Reports, 1997,16:594-599
174. Thinh NT, Takagi H, Yashima S. Cryopreservation of in vitro grown shoot tips of banana (Musa spp) by vitrification method[J]. Cryo-Letters, 1999, 20: 163-174
175. Touchell D H, Ching V L, Tsai C J. Cryopreservation of embryogenic cultures of Picer mariana (black spruce) using vitrification[J]. Plant Cell Reports.2002,21:1 18-124
176. Touchell D H, Dixon K W. Cryopreservation for the conservation of native Australian endangered plants, in: M.N. Normah, M.K. Narimah, M.M. Clyde (Eds.), Plant Biotechnology Laboratory, University Kebangsaan Malaysia, Kuala Lumpur, Malaysia, 1996,169-180
177. Towill L E. Cryopreservation of isolated mint shoot tips by vitrification [J]. Plant Cell Reports. 1990,9:178-1180
178. Towill L E. Cryopreservation of Plant Germplasm II. p4.2002
179. Towill LE, Jarret RL. Cryopreservation of sweet potato (Ipomoea batatas [L.] Lam.) shoot tipsby vitrification[J]. Plant Cell Reports. 1992,11:175-178
180. Tsukazaki, H. Mii, M. K. Tokuhara, Ishikawa, K. Cryopreservation of Doritaenopsis suspension culture by vitrification[J]. Plant Cell Reports.2000,19:1160-1164
181. Turner S R, Senaratna T, Bunn E, et al. Cryopreservation of shoot tips from six endangered australian species using a modified vitrification protocol[J]. Annals of Botany.2001,87:371-378
182. Kuriyama A, Watanabe K, Ueno S, et al. Inhibitory effect of ammonium ion on recovery of cryopreservation rice cells. Plant Science, 1990,64:231-235
183. Turner S, Senaratna T, Touchell D, et al. Stereochemical arrangement of hydroxyl groups in sugar and polyalcohol molecules as an important factor in effective cryopreservation. Plant Science. 2001,160:489-497
184. Turner S, Siegfried LK, Eric B, et al. Genetic fidelity and viability of Anigozanthos viridis following tissue culture, cold storage and cryopreservation. Plant Science. 2001, 161: 1099-1106
185. Uragami, A. Sakai, A. Nagai, M. Takahashi, T. Cryopreservation by vitrification of cultured cells and somatic embryo from mesophyll tissue of asparagus [J]. Acta horticulturae. 1990 July,(271):109-116
186. Uragami A, Sakai A, Nagai M, Takahash T. Survival of cells and somatic embryos of Asparagus officinalis cryopreserved by vitrification[J]. Plant Cell Reports. 1989,8: 418-421
187. Vandenbussche B, Weyens G, De Proft M. Cryopreservation of in vitro sugar beet (Beta vulgaris L.) shoot tips by a vitrification technique [J]. Plant Cell Reports. 2000,19:1064-1068
188. Vidal N, Sanchez C, Jorquera L, et al.Cryopreservation of chestnut by vitrification of intro-grown shoot tips [J]. In Vitro Cellular & Development Biology. 2005,41: 63-68
189. Volk G M, Richards K, Preservation methods for Jerusalem artichoke cultivars[M]. HortScience: a publication of the American Society for Horticultural Science. 2006 Feb,41(1):80-83
190. Wang Q Ch , Mawassi M, Li P,et al. Elimination of grapevine virus A (GVA) by cryopreservation of in vitro-grown shoot tips of Vitis vinifera L[J].Plant Science.2003,165:321-327
191.Watanabe K and Steponkus P L. Vitrification of Oryza sativa L. cell suspensions[J].Cryo-letters.1995,16 (5) : 255-262
192. Wu Y J, Huang X L, Li Q Zh, et al. Induction and cryopreservation of embryogenic cultures from nucelli and immature cotyledon cuts of mango (Mangifera indica L. var. Zihua). Plant cell reports. 2007, 26(2):161-168
193. Yamada AK. Cryopreservation of in vitro-grown apical meristems of lily by vitrification. Plant Cell Tissue Organ Culture. 1995, (41): 237-241
194. Yamada T, Sakai A, Matsumura T, et al. Cryopreservation of apical meristems of white clover (Trifolium repens L.) by vitrification[J]. Plant Science. 1991, (78): 81-87
195. Vidal N, Sanchez C, Jorquera L, et al. Cryopreservation of chestnut by vitrification of in vitro-grown shoot tips. In Vitro Cell. Dev. Biol.-Plant,2005,41:63-68
196. Yoshimatsu K, Yamaguchi H, Shimomura K. Traits of Panax ginseng hairy roots after cold storage and cryopreservation [J]. Plant Cell Reports, 1996, 15: 555-560
197. Zhao M A, Xhu Y Z.,Dhital S P, et al. An efficient cryopreservation procedure for potato (Solanum tuberosum L.) utilizing the new ice blocking agent, Supercool×1000[J]. Plant cell reports.2005, 24(8):477-481
2．艾鹏飞，卢利平．桔梗试管苗苇尖玻璃化法超低温保存及植株再生[J]．中草约，2006，37(9)：1409-1412
3．艾鹏飞，罗正荣．柿休眠芽茎尖玻璃化法超低温保存及植株再生[J]．中国农业科学，2003，36(5)：553-556
4．敖妍，鲁韧强，潘青华．扶芳藤快速扩繁试验[J]．林业科技开发，2006，21(1)：36-39
5．蔡小宁，陈舒泛，陈俊，等．小球藻的玻璃化超低温保存[J]．植物生理学通讯，2004，40(5)：599-601
6．蔡祖国，徐小彪，赵一鹏，等．猕猴桃茎尖超低温保存过程中超微结构观察[J]．西北植物学报，2006，26(8)：1600-1604
7．陈勇，王君晖，黄纯农．铁皮石斛种质资源的玻璃化法超低温保存[J]．浙江大学学报(农业与生命科学版)，2001，27(4)：436-438
8．陈黎．复方扶芳藤合剂抗大肠癌术后化疗白细胞减少疗效观察[J]．广西中医药，2001，24(5)：297-298
9．陈品良．植物组织培养物的超低温保存[J]．武汉植物研究，1989，7(4)：390-398
10．陈书安，王晓东，赵兵，等．水母雪莲愈伤组织超低温保存条件的初探[J]．过程工程学报，2002，2(6)：539-543
11．陈勇，陈娴婷，王君晖．瓯柑愈伤组织的玻璃化法超低温保存研究[J]．浙江大学学报(理学版)，2004，31(2)：197-201
12．陈勇，王君晖，黄纯农．胡萝卜悬浮培养细胞和原生质体的玻璃化法超低温保存[J]．浙江大学学报(理学版)，2002，29(1)：94-98
13．邓家刚．复方扶芳藤合剂异病同治举偶[J]．广西中医药，2000，23(3)：47-48
14．董丽，黄亦工，贾麦娥，等．北京园林主要常绿阔叶植物抗冻性及其测定方法[J]．北京林业大学学报，2002，24(3)：70-73
15．高建钰，马晶，樊明瑞，等．扶芳藤营养袋育苗试验[J]．山西林业科技，2005，1：12-14
16．葛锋，王晓东，赵兵，等．肉从蓉愈伤组织的超低温保藏方法[J]．过程工程学报，2006，6(5)：794-798
17．关雪莲，马清水，张宁，等．大叶黄杨(Euonymus japonicus)和扶芳藤(Euonymus fortunei)耐寒生理指标的变化．北京农学院学报，2006，7：29-33
18．郭燕霞，刘玉军．长鞭红景大悬浮培养细胞的玻璃化法超低温保存研究[J]．西北植物学报，2006，26(8)：1605-1611
19．胡明珏，王君晖．拟南芥悬浮细胞系的玻璃化法超低温保存[J]．细胞生物学杂志，2004，26(1)：81-84．
20．黄纯农，王君晖，颜秋生，等．大麦胚性悬浮细胞系的快速建立及其超低温保存[J]．中国农业科学，1995，28(3)：21-27
21．黄纯农，颜秋生．玻璃化法液氮冻存大麦和水稻的胚性悬浮细胞[J]．杭州大学学报：自然科学版，1994，21(1)：114-115
22．简令成．低温生物学与植物种质的K期保存[J]．植物学通报，1988，5(2)：65-68
23．缴丽莉，路丙社，白志英，等．四种园林树木抗寒性的比较分析[J]．园艺学报，2006，33(3)：667-670
24．金万梅，尹淑萍，鲁韧强，等．扶芳藤组织培养再生体系的建立[J]．植物生理学通讯，2005，41(1)：27-30
25．李广武、郑从义等．低温生物学[M]．长沙：湖南科技出版社，1998
26．李合生．植物生理生化实验原理和技术．北京：高等教育出版社，2000，258-260
27．李吉跃．太行山区主要造林树种耐早特性的研究[D1]．北京林业大学博士学位论文，1990
28．李锦树，王洪春，王文英．1983．干旱对玉米叶片细胞膜透性及膜脂的影响．植物生理学通讯，1983，9：223-228
29．李明军，洪森荣，徐鑫，等．怀山药种质资源的玻璃化法超低温保存[J]．作物学报，2006，2，288-292
30．李鹏，李航．扶芳藤中的三萜化合物研究[J]．中国药学杂志，2000，35(12)：847-848
31．李雪芹，徐礼根，金松恒，马博英．4种草坪草叶绿素荧光特性的比较[J]．园艺学报，2006，33(1)：164-167
32．林田，刘灶长，李天菲，等．红花石蒜茎尖的玻璃化超低温保存[J]．植物生理学通讯，2006，42(6)：1063-1066
33．刘长利，甘草抗旱特性的初步研究，河北农业大学，硕士学位论文，2002
34．刘峰，颜秋生．水稻胚性悬浮细胞玻璃化冻存中的超微结构变化[J]．中国水稻科学，1998，12(1)：17-20
35．刘小攀，姜超．利用槐荫扦插扶芳藤[J]．林业实用技术，2003，5：24
36．刘燕，高荣孚，周晓刚．玻璃化超低温保存中拟南芥幼苗细胞膜ATPase的变化[J]．北京林业大学学报，2001，23(5)：1-5
37．刘燕．拟南芥幼苗玻璃化超低温保存研究．北京林业大学，博士论文，2000
38．刘云国，王晓云．苹果种质资源玻璃化法超低温保存技术[J]．山东农业大学学报(自然科学版)，2002，33(1)：32-36
39．刘云国．苹果种质资源玻璃化法超低温保存技术及其遗传变异分析．山东农业大学，博士论文．
40．罗俊，张木清，林彦铨，张华，陈如凯，甘蔗苗期叶绿素荧光参数与抗旱性关系研究[J]．中国农业科学 2004，37(11)：1718-1721
41．马锋旺，李嘉瑞．杏原生质体的超低温[J]．园艺学报．1998，25(4)：329-332
42．梅俊学．逆温下发菜脯氨酸含量及膜透性的变化与含水量的关系．山东师大学报(自然科学版)，15(2)：178-181
43．孟庆辉，潘青华，鲁韧强，等．4个品种扶芳藤茎叶解剖结构及其与抗旱性的关系[J]．农业基础科学，2006，22(4)：138-142
44．潘青华，宋婉，鲁韧强，等．扶芳藤种质资源及变异研究[J]．北京林业大学学报，2004，26(2)：58-62
45．潘青华．扶芳藤遗传多样性研究及抗逆性选择．北京林业大学，2003，博士论文
46．彭志红，彭克勤，胡家金，萧浪涛．渗透胁迫下植物脯氨酸积累的研究进展[J]．中国农学通报，2002，18(4)：80-83。
47．曲宝军，李大亮．扶芳藤对不同依附体的攀援能力试验[J]．山东林业科技，2001，135(4)：35
48．邵建柱．苹果等果树种质资源的离体保存研究．华中农业大学，博士论文，2003
49．史燕山，李嘉，李长槐，等．生长素对花叶扶芳藤扦插成活及苗质的影响[J]．天津农业科学，2004，10(1)：4-6
50．苏文华，张先辉，王崇云，等．短葶飞蓬光合生理生态的初步研究[J]．云南大学学报，2001，23：142-145
51．唐连顺，李广敏．水分胁迫下玉米叶肉细胞超微结构的变化及其与膜脂过氧化伤害的关系[J]．植物学报，1994，36(增刊)：43-49
52．王和祥，么秀文．木本地被植物引种栽培的研究[J]．园林科技通讯，1997，28：2-10
53．王建 药用植物扶芳藤的组织培养．广西中医学院学报，2004，7(2)：62-63
54．王君晖，严庆丰，黄纯农．大麦幼穗的玻璃化法超低温保存及冻后植株再生[J]．植物学报，1996，38(9)：730-734
55．王君晖，张毅翔，刘峰，等．铁皮石斛种子、原球茎和类原球茎体的超低温保存研究[J]．园艺学报，1999，26(1)：59-61
56．王君晖，郑勇．水稻胚性悬浮细胞的玻璃化超低温保存和可育植株再生[J]．科学通报，1996，41(22)：2081-2084
57．王君辉，黄纯安．玻璃化法-园艺作物茎尖和分生组织超低温保存的新途径[J]．园艺学报，1994，21(3)：277-282．
58．王茂良，赵粱军，任桂芳，等．扶芳藤再生体系的建立[J]．园艺学报，2004，31(2)：241-244
59．王艳军，李锡香，向长萍，等．大蒜茎尖玻璃化法超低温保存技术研究[J]．园艺学报，2005，32(3)：507-509
60．王雁，马武昌．扶芳藤、紫藤等7种藤本植物光能利用特性及耐荫性比较研究[J]．林业科学研究，2004，17(3)：305-309
61．王永林．马铃薯茎尖超低温保存研究[J]．甘肃农业科技，2006(10)：3-5
62．王子成，邓秀新．柑橘体细胞杂种超低温保存后的植株再生．园艺学报，2004，31(2)：215-216
63．王子成，邓秀新．玻璃化法超低温保存柑桔茎尖及植株再生[J]．园艺学报，2001，28(4)：301-306
64．王家福，刘月学．枇杷茎尖二步玻璃化法超低温保存的研究[J]．植物资源与环境学报，2006，15(2)：75-76
65．吴黎明，曾继吾，彭抒昂，等．香蕉茎尖超低温保存过程中的细胞超微结构观察[J]．分子细胞生物学，2006，39(6)：563-567
66．吴庆华，李春霞．扶芳藤直接扦插栽培初探[J]．中国野生植物资源，2005，24(3)：59
67．伍小燕，黄莹娟．扶芳藤对血栓形成及凝血系统的影响[J]．西北药学杂志，1997，12(1)：19-20
68．项黎新，邵健忠．衣藻细胞坡璃化超低温保存技术的研究[J]．细胞生物学杂志，2001，110-113
69．肖洁凝，黄学林．茎尖利芽的超低温保存[J]．生物工程进展，1999，19(5)：46-51
70．谢金鲜，林启云．扶芳藤对心血管作用的试验研究[J]．广西中医药，1999，51-53
71．徐刚标．植物种质资源离体保存研究进展[J]．中南林学院学报，2000，20(4)：81-87
72．薛建平，张爱民，柳俊，等．玻璃化法超低温保存地黄茎尖[J]．农业生物技术学报，2003，11(4)：430-431
73．严庆丰，黄纯农．植物组织和细胞的玻璃化冻存研究[J]．细胞生物学杂志，1994，16(3)：117-122
74．阎秀峰，孙国荣，肖玮．星星草光合蒸腾特性的季节变化[J]．植物研究，1996，16(3)：340-345
75．殷晓辉，舒理慧，郑从义，等．野生稻愈伤组织的超低温保存和冻后再生植株的形成[J]．武汉植物学研究，1996，14(3)：247-252
76．喻方圆，徐锡增．植物逆境生理研究进展．世界林业研究，2003，16(5)：6-11
77．曾继吾，易干军，张秋明，等．番术瓜茎尖超低温保存过程中的细胞超微结构[J]．园艺学报，2005，32(1)：15-19
78．曾继吾，易干军，张秋明，等．果树种质资源离体保存研究进展[J]．果树学报，2002，19(5)：302-306
79．翟发林，丁青龙，张汉民．扶芳藤化学成分研究[J]．南京军医学院学报，2001，23(4)：221-223，226
80．张德舜，刘红权．从种常绿阔叶树种抗寒性的研究[J]．园艺学报，1994，21(3)：283-287
81．张明生，谢波，谈锋，张启堂．甘薯可溶性蛋白、叶绿素及ATP含量变化与品种抗旱性关系的研究[J]．中国农业科学，2003，36(1)：13-16
82．张丕方，倪德祥，包慈华．植物组织培养与繁殖上的应用[M]。上海教育出版社，1985：45-48
83．张守梅，李建国，陈厚彬，等．香蕉茎尖滴冻法保存中细胞的超微结构变化[J]．园艺学报，2006，33(4)：709-713
84．张守仁．叶绿素荧光动力学参数的意义及讨论．植物学通讯，1999，16(4)：444-448
85．张演义，孙仲序，刘建强等．超低温保存果树种质资源研究进展[J]．山东林业科技，2002，138(1)：45-47
86．张玉进，张兴国，庞杰，等．魔芋茎尖玻璃化冻存研究[J]．作物学报，2001，27(1)：97-102
87．张玉进、张兴国，刘佩瑛，等．植物茎尖的玻璃化冻存研究[J]．武汉植物学研究，1999，17(增刊)：78-82
88．张玉芹，李锡香，马庆．实用百合种质的玻璃化法超低温保存技术初探[J]．中国蔬菜，2004(4)：11-13
89．赵黎芳，张金政，张启翔，等．水分胁迫下扶芳藤幼苗保护酶活性和深透调节物质的变化[J]．植物研究，2003，23(4)：437-442
90．赵黎芳，张金政，张启翔，等．盐和水分预处理对扶芳藤幼苗抗寒性的影响[J]．植物研究，2004，24(3)：313-316
91．赵艳华，吴雅琴，李春敏．梨离体茎尖超低温保存方法的比较研究[J]．河北农业科学，2004，8(4)：93-95
92．赵艳华，吴雅琴．桃离体茎尖的超低温保存及植株再生[J]．园艺学报，2006，33(5)：1042-1044
93．赵艳华，吴永杰，周明德．马哈利樱桃离体茎尖超低温保存的研究[J]．园艺学报，1999，26(6)：402-403
94．郑国生，何秀丽．夏季遮荫改善大田牡丹叶片光合功能的研究[J]．林业科学，2006，42(4)：27-32
95．周小梅，王国英，敖光明，等．玉米组培材料的玻璃化法超低温保存及冻后植株再生[J]．农业生物技术学报，2001，9(4)：355-358
96．周雪玲，付超，陈奇凌．扶芳藤组织培养技术研究[J]．新疆农业科学，2006，1：47
97．朱红梅，钟鸣，黄琳芸，等．扶芳藤及其提取物有关药理作用的实验研究[J]．中国中医药科技，2000，7(3)：170
98．朱西存，卢胜西，时鑫．扶芳藤特性及扦插繁育技术[J]．山东林业科技，2004，152(3)：42
99. Ahn Y H, and Sakai A. Survival by vitrification of Sendtanthma grandiforum L. shoot tips for cryopreservation. Horticultural Science, 1994, 35(5): 499-506
100. Angelika SM, Schumacher HM, Gunda MW. Long-term storage of old patato varieties by cryopreservation of shoot-tips in liquid nitrogen. Plant Genetic Resource Newsletter, 1997, (111): 19-24
101. Arnon D I. Copper enzymes in isolated chloroplasts. Polyphenoloxidase in Beta vulagris. Plant Physiology, 1949, 24:1-15
102. Baek H J, Kim H H, Cho E G. et al. Importance of explant size and origin and of preconditioning treatments for cryopreservation of garlic shoot apices by vitrification[J]. Cryo-letters. 2003,24(6):381-388
103. Beadle C L, Ludlow M M, Honeysett J L. In:Hall, E. O., J. M. O. Scurlock & H. R. Bolhar Norderkampf eds. Photosynthesis and production in a changing environment. A field and laboratory manual. 1993, London :Chapman & Hall.
104. Ben G Y, Osmond C B and Sharkey T D. Comparisons of photosynthetic responses of Xanthium strumarium and Helianthus annuus to chronic and acute water stress in sun and shade[J]. Plant Physiology, 1987, 84:476-482.
105. Benelli C, De Carlo A, Lambardi M, et al. Vitrification of shoot tips, nodal segments and embryogentic tissue of olive (Olea europaea L.) for germplasm cryopreservation[J]. Acta horticulturae. 2001,560:137-140
106. Bowler C, Montagu M V, Inze D. Superoxide dismutase and stress tolerance[J]. Annual Review of Plant Physiology and Plant Molecular Biology, 1992,43:83-116.
107. Channuntapipat C, Collins G, Bertezzi T, et al. Cryopreservation of in vitro almond shoot tips by vitrification[J]. Journal of horticultural science & biotechnology. 2000,75(2):228-232
108. Charoensub R, Phansiri S, Yongmanitchai W, et al. Routine cryopreservation of in vitro-grown axillary apices of cassava (Manihot esculenta Crantz) by vitrification: importance of a simple mononodal culture[J]. Scientia Horticulturea.2003,98: 485-492
109. Cornic G J and Briantais J M. Partitioning of photosynthetic CO2 and O2 reduction in a C3 leaf (Phaseolus vulgaris L.)at different CO2 concentrations during drought stress[J]. Planta 1991,183:178-184
110. Escobar R H, Mafia G, Roca W M. A methodology for recovering cassava plants from shoot tips maintained in liquid nitrogen. Plant Cell Reports. 1997,16,474-478
111. Crowe J H, Crowe L M. Carpenter J ￥,et al. Interaction of sugars with membranes. Biochim. Biophys. Acta, 1989 947:367-384
112. Crowe J H,Carpenter J F,Crowe L M,et al. Are freezing and dehydration similar stress vectors? A comparison of modes of interaction of stailizing solutes with biomolecules. Cryobilogy, 1990,27:219-231
113. Crowe L M, Crowe J H. Stabilisation of membranes in anhydrobiotic organisms. In: Leopld A C (ed) Memberanes, metabolism, and dry organisms. Comstock, Cornell University, New York, 1986,210-230
114. DeVerno L L, Park Y S, Bonga J M, et al. Somaclonal variation in cryopreserved embryogenic clones of white spruce. Plant Cell Reports, 1999, 18(11):948-953
115. Engelmann F, Aguilar M E, Dambier D, et al. Advanteges of cryopreservation of cell suspensions and embryogenic calli for citrus breeding proprammers. Fruit, 1994,49(1):23-30
116. Halmagyi & Pinker. Plant regeneration from Rosa shoot tips cryopreserved by a combined droplet vitrification method [J]. Plant cell, Tissue and Organ culture. 2006,84(2): 145-153
117. Halmagyi A, Fisher-Kluver G, Mix-Wagner, et al. Cryopreservation of Chrysanthemum morifolium (Dendranthema grandiflora Ramat.) using different approaches[J]. Plant cell reports.2004,22(6):371 -375
118. Helliot B, Panis B, Poumay Y, et al. Cryopreservation for the elimination of cucumber mosaic and banana streak viruses from banana (Mus aspp.) Plant Cell Reports. 2002,20:1117-1122
119. Helliot B, Swennen R, Poumay Y, et al. Ultrastructural changes associated with cryopreservation of banana (Musa spp.) highly proliferating menstems. Plant Cell Reports, 2003,21 (7): 690-698
120. Hely M. Haggman, Leena A. Ryynanen, Tuija S. et al. Cryopreservation of embryogenic cultures of Scots pine[J]. Plant cell, tissue and organ culture.1998(54):45-53
121. Hirai D and Sakai A.Cryopreservation of in Vitro-grown meristems of patotato(Solanum tuberosum L.) by encapsulation-vitrification[J].Patato Research. 1999,42:153-160
122. Hirano T, Godo T, Mii M. et al. Cryopreservation of immature seeds of Bletilla striata by vitrification[J]. Plant cell reports. 2005,23(8):534-539
123. Hubscher S L, Touchell D H,C-J Tsai. Cryopreservation of transgenic populus tremuloides using a modified vitrification protocol. In vitro Cellular & Developmental Biology; Spring 2004; 40, ProQuest Biology Journals, pg. 73
124. Ishikawa K, Harata K, Mii M, et al. Cryopreservation of zygotic embryos of a Japanese terrestrial orchid (Bletilla striata) bsy vitrification[J]. Plant Cell Reports, 1997, 16 (11): 754-757
125. Ishikawa K,Harata K,Sakai A, et al. Cryopreservation of zygotic embryos of a Japanese terrestrial orchid (Blettilla striata) by vitrification[J], Plant Cell Reports. 1997,16(11):754-757
126. Ishikawa M, Tandon P, Suzuki M, et al. Cryopreservation of bromegrass (Bromus inermis Leyss) suspension cultured cells using slow prefreezing and vitrification procedures[J]. Plant Science, 1996, 120: 81-88
127. Ishikawa M,Suzuki M,Kishimoto T, et al. Effect of growth phase on survival of bromegrass suspension cells following cryopreservation and abiotic stresses[J] .Annals of botany. 2006, 97(3):453-459
128. Jung D W, Sung C K, Touno K, et al. Cryopreservation of Hyoscyamus niger adventitious roots by vitrification. J. Plant Physiology, 2001, 158: 801-805
129. Kanchit T. Cryopreservation of seeds of Thai orchid (Doritis pulcherrima Lindl.) by vitrification[J]. Cryo letter.2000,21:237-244
130. Kohmura H, Ikeda Y, Sakai A. Cryopreservation of apical meristems of Japanese shallot (Allium wakegi A.) by vitrification and subsequent high plant regeneration[J]. Cryo-letters. 1994,15(5):289-298
131. Kohmura H, Saka i A, Chokyu S, et al. Cryopreservation of in vitro-cultured multiple bud clusters of asparagus (Asparagus officinalis L. cv. Hiroshimagreen (2n=30)) by the techniques of vitrification[J]. Plant Cell Reports, 1992, 11: 433-437
132. KuranukiY, Sakai A, Cryopreservation of intro-grown shoot tips of tea (Camellia sinensis)by vitrification[J]. Cryo-letters. 1995,16(6):345-351
133. Lambardi M, Fabbri A, Caccavale A.Cryopreservation of white poplar (Populus alba L.) by vitrification of in vitrogrown shoot tips[J]. Plant Cell Reports.2000, 19:213-218
134. Langis R, Schnabel-Preikstas B, Earle ED, Steponkus P L. Cryopreservation of Brassica compestris cell suspensions by vitrification[J]. Cryo-letters, 1989, 10: 421-428
135. Langis R, Stepinkus P L. Vitrification of ioslated rye protoplasts: protection against dehydration injury by ethylene glycol[J].Cryo-letters.l991,12(2):107-112
136. Langis R, Steponkus P L. Cryopreservation of rye protoplasts by vitrification[J]. Plant Physiology[J].1990, 92, 666-671
137. Leunufna S, Keller E R J. Investigating a new cryopreservation protocol for yams (Dioscorea spp.) [J].Plant Cell Reports.2003,21:1159-1166
138. Lichtenthaler H K . Application of chlorophyll fluorescence in research stress physiology [M],hydrobiology and remote sensing Kluwer Academic Punlishers,Dordrecht, 1991,253-258
139. Liu H Yu, W Dai, J Gong, et al. Cryopreservation of protoplasts of the alga Porphyra yezoensis by vitrification[J]. Plant science. 2004,166(1): 97-102
140. Liu Y G, Wang X Y, Liu L X. Analysis of genetic variation in surviving apple shoots following cryopreservation by vitrification[J]. Plant science. 2004 Mar,166(3):677-685
141. Luo J, Reed B M. Abscisic acid-responsive protein, bovine serum albumin, and praline pretreatments improve recovery of in vitro currant shoot-tip meristems and callus cryopreserved by vitrification[J] .Cryopreservation. 1997, 34:240-250
142. Luyet,B.J. Biodymanica.l937,1:1-14
143. MacFarlane D R,Devitrification in grass forming aqueous solutions,Cryobiology,1986,23: 230-244
144. Maruyama E, Kinoshita I,Ishii K,Ohaba K. and Sakai A. Gemplasm convervation of Guazuma crinita,a useful tree in peru-Amazon,by the cryopreservation of in vitro-cultured multiple bud clusters[J].Plant Cell Tissue and Organ Culture.l997,43(3):161-163
145. Matsumoto T, Mochida K, Itamura H, et al. Cryopreservation of persimmon (Diospyros kaki Thunb.)by vitrification of dormant shoot tips[J].Plant Cell Reports.2001,20:398-402
146. Matsumoto T, Nako Y, Takahashi C,et al. Induction of in vitro cultured masses of shoot primordia of hybrida satatice and its cryopreservation by vitrification[J]. HortScience. 1997,32(2):309-311
147. Matsumoto T, Sakai A, Yamada K Cryopreservation of in vitro-grown apical meristems of lily by vitrification. Plant Cell Tissue Organ Culture., 1995, 41: 237-241
148. Matsumoto T, Sakai A, Yamada K. Cryopreservation of in vitro-grown apical meristems of wasabi (Wasabia japonica) by vitrification and subsequent high plant regeneration[J]. Plant Cell Reports. 1994,13:442-446
149. Matsumoto T, Sakai A. Cryopreservation of axillary shoot tips of in vitro-grown grape(Vitis) by a two-step vitrification protocol[J]. Euphytica: international journal of plant breeding.2003,131 (3):299-304
150. Matsumoto T, Takahashi C, Sakai A, et al. Cryopreservation of in vitro-grown apical meristems of hybrid statice by three different procedures. Scientia Horticture., 1998, 76: 105-114
151. Na H Y, Kondo K. Cryopreservation of tissue-cultured shoot primordia from shoot apices of cultured protocorms in Vanda pumila following ABA preculture and desiccation[J] .Plant Science. 1996,118:195-201
152. Niino T, Sakai A, Yakuwa H, et al. Cryopreservation of in vitro-grown shoot tips of apple and pear by vitrification.Plant Cell[J] .Tissue and Organ Culture. 1992,28:261-266
153. Niino T,Tashiro K, Suzuki M. et al. Cryopreservation of in vitro grown shoot tips of herry by one-step vitrification[J]. cientia horticulturae.l997,July,70(2-3):155-163
154. Nishizawa S, Sakai A, AmanoY. Cryopreservation of Asparagus (Asparagus officinalis L.) embryogenic suspension cell and subsequent plant regeneration by vitrification. Plant Science 1993,91:67-73
155. Panis B, Piette B, Swennen R. Droplet vitrification of apical meristems: a cryopreservation protocol applicable to all Musaceae[J].Plant science. 2005,168(1):45-55
156. Panis B, Totté N, Nimmen K V, et al. Cryopreservation of banana (Musa spp.) meristem cultures after preculture on sucrose[J]. Plant Science. 1996,121:95-106
157. Pennycooke JC, Towill LE. Cryopreservation of shoot tips from in vitro plants of sweet potato (Ipomoea batatas L.) by vitrification[J]. Plant Cell Reports.2000, 19:733-737
158. Polge C. and Smith A.U. Reviaval of spermatozoa after vitrifiation and dehydration at low temperature[J]. Nature. 1949,164:666
159. Rall W F and Fahy G M,Nature.l985,313:573-575
160. Reed B M and Yu X. Cryopreservation of in vitro-grown gooseberry and currant meristems. CryoLetters. 1995,16: 131-136
161.Reed BM, Dumet D, Denoma JM. Validation of cryopreservation protocols for plant germplasm conservation: a pilot study using Ribes L [J]. Biodiversity and Conservation, 2001, 10: 939-949
162. Reinhoud P J, Schrijinemarkers E W M, Iren F.et al. Vitrification and heat-shock treatment improve cryopreservation of tobacco cell suspensions compared to two-step freezing[J]. Plant cell, tissue and organ culture.1995,42(3):261-267
163. Sakai A, Kobayashi S, Oiyama I. Cryopreservation of nucellar cells of navel orange (Citrus sinensis var. brasiliensis Tanaka) by vitrification. Plant Cell Reports, 1990, 9: 30-33
164. Sakai A, Niino T, Yamada T, Kohmura H. Cryopreservation by vitrification of mulberry meristematic callus of white clever and multiple bud clusters of asparagus. Cryobiology, 1992, 29 (6): 747-748
165. Ahuja S, Mandal B B, Dixit S, et al. Molecular, phenotypic and biosynthetic stability in Dioscorea floribunda plants derived from cryopreserved shoot tips[J] .Plant Science.2002,163:971-977
166. Sarkar D,Naik P S. Cryopreservation of shoot tips of tetraploid potato (Solarium tuberosum L.) clones by vitrification[J]. Annals of Botany. 1998,82:455-461
167. Schafer-Menuhar A,Schumarcher H and Wangner G. Long—term storage of old potato varieties by cryopreservation of shoot-tips in liquid nitrogen[J]. Plant Gen. Res. News Lett. 1997,111:19-24
168. Scheuermann,R.,K,Bichler,T. Stuhlfauth and H. Fork. Simultaneous gas exchange and fluorescence measurements indicate differences in the response of sunflower,bean and maize to water stress[J].Photosynthesis Research. 1991,27:189-197
169. Sellferheld M J, Stushnoff C, Fitzpatrick J, et al. Cryopreservation of dormant buds after cold-tender taxa using a modified vitrification procedurer, Hortscicence,1992,27(12):1262
170. Skirvin R M, McPheeters K,Norton M. Sources and frequency of somaclonal variation[J]. Horscience. 1994,29:1232-1235
171. Steponkus P L, Langis R, Fujikawa S. Cryoprservation of plant tissue by vitrification. In: Advances in Low-temperature Biology, edited by Steponkus P.L. JAI press, Ltd., London, 1992, 1: 1-6
172. Tsukazaki H, Mii M, Tokuhara K, et al. Cryopreservation of Doritaenopsis suspension culture by vitrification[J]. Plant cell report.2000, 19(12):1160-1164
173. Takagi H, Thinh NT, Islam OM, et al. Cryopreservation of in vitro-grown shoot tips of taro (Colocasia esculenta (L.) Schott) by vitrification. 1.Investigation of basic conditions of vitrification procedure[J].Plant Cell Reports, 1997,16:594-599
174. Thinh NT, Takagi H, Yashima S. Cryopreservation of in vitro grown shoot tips of banana (Musa spp) by vitrification method[J]. Cryo-Letters, 1999, 20: 163-174
175. Touchell D H, Ching V L, Tsai C J. Cryopreservation of embryogenic cultures of Picer mariana (black spruce) using vitrification[J]. Plant Cell Reports.2002,21:1 18-124
176. Touchell D H, Dixon K W. Cryopreservation for the conservation of native Australian endangered plants, in: M.N. Normah, M.K. Narimah, M.M. Clyde (Eds.), Plant Biotechnology Laboratory, University Kebangsaan Malaysia, Kuala Lumpur, Malaysia, 1996,169-180
177. Towill L E. Cryopreservation of isolated mint shoot tips by vitrification [J]. Plant Cell Reports. 1990,9:178-1180
178. Towill L E. Cryopreservation of Plant Germplasm II. p4.2002
179. Towill LE, Jarret RL. Cryopreservation of sweet potato (Ipomoea batatas [L.] Lam.) shoot tipsby vitrification[J]. Plant Cell Reports. 1992,11:175-178
180. Tsukazaki, H. Mii, M. K. Tokuhara, Ishikawa, K. Cryopreservation of Doritaenopsis suspension culture by vitrification[J]. Plant Cell Reports.2000,19:1160-1164
181. Turner S R, Senaratna T, Bunn E, et al. Cryopreservation of shoot tips from six endangered australian species using a modified vitrification protocol[J]. Annals of Botany.2001,87:371-378
182. Kuriyama A, Watanabe K, Ueno S, et al. Inhibitory effect of ammonium ion on recovery of cryopreservation rice cells. Plant Science, 1990,64:231-235
183. Turner S, Senaratna T, Touchell D, et al. Stereochemical arrangement of hydroxyl groups in sugar and polyalcohol molecules as an important factor in effective cryopreservation. Plant Science. 2001,160:489-497
184. Turner S, Siegfried LK, Eric B, et al. Genetic fidelity and viability of Anigozanthos viridis following tissue culture, cold storage and cryopreservation. Plant Science. 2001, 161: 1099-1106
185. Uragami, A. Sakai, A. Nagai, M. Takahashi, T. Cryopreservation by vitrification of cultured cells and somatic embryo from mesophyll tissue of asparagus [J]. Acta horticulturae. 1990 July,(271):109-116
186. Uragami A, Sakai A, Nagai M, Takahash T. Survival of cells and somatic embryos of Asparagus officinalis cryopreserved by vitrification[J]. Plant Cell Reports. 1989,8: 418-421
187. Vandenbussche B, Weyens G, De Proft M. Cryopreservation of in vitro sugar beet (Beta vulgaris L.) shoot tips by a vitrification technique [J]. Plant Cell Reports. 2000,19:1064-1068
188. Vidal N, Sanchez C, Jorquera L, et al.Cryopreservation of chestnut by vitrification of intro-grown shoot tips [J]. In Vitro Cellular & Development Biology. 2005,41: 63-68
189. Volk G M, Richards K, Preservation methods for Jerusalem artichoke cultivars[M]. HortScience: a publication of the American Society for Horticultural Science. 2006 Feb,41(1):80-83
190. Wang Q Ch , Mawassi M, Li P,et al. Elimination of grapevine virus A (GVA) by cryopreservation of in vitro-grown shoot tips of Vitis vinifera L[J].Plant Science.2003,165:321-327
191.Watanabe K and Steponkus P L. Vitrification of Oryza sativa L. cell suspensions[J].Cryo-letters.1995,16 (5) : 255-262
192. Wu Y J, Huang X L, Li Q Zh, et al. Induction and cryopreservation of embryogenic cultures from nucelli and immature cotyledon cuts of mango (Mangifera indica L. var. Zihua). Plant cell reports. 2007, 26(2):161-168
193. Yamada AK. Cryopreservation of in vitro-grown apical meristems of lily by vitrification. Plant Cell Tissue Organ Culture. 1995, (41): 237-241
194. Yamada T, Sakai A, Matsumura T, et al. Cryopreservation of apical meristems of white clover (Trifolium repens L.) by vitrification[J]. Plant Science. 1991, (78): 81-87
195. Vidal N, Sanchez C, Jorquera L, et al. Cryopreservation of chestnut by vitrification of in vitro-grown shoot tips. In Vitro Cell. Dev. Biol.-Plant,2005,41:63-68
196. Yoshimatsu K, Yamaguchi H, Shimomura K. Traits of Panax ginseng hairy roots after cold storage and cryopreservation [J]. Plant Cell Reports, 1996, 15: 555-560
197. Zhao M A, Xhu Y Z.,Dhital S P, et al. An efficient cryopreservation procedure for potato (Solanum tuberosum L.) utilizing the new ice blocking agent, Supercool×1000[J]. Plant cell reports.2005, 24(8):477-481