Effect Judgement Of Euonymus Fortunei Plants Derived From Cryopreserved Shoot Tips By Vitrification

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.