【作者】 王贞；

【导师】 刘燕；

【作者基本信息】 北京林业大学 ， 园林植物与观赏园艺， 2007， 博士

【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。更多还原

【Abstract】 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 CO₂ compensation point, CO₂ 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（F₀, Fm,Fv, Fv/F₀, 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.更多还原