| There are12807kinds of traditional Chinese medicine resources in china,among which11146kinds are of medicinal plants, accounting for87.03%. At presentnearly2100species in the endangered state, using the field planting to save quantityof germplasm resources, not only a lot of pressure on the soil, but also on manpowerand material resources, are vulnerable to natural disasters. New cryopreservationtechnique developed in the1970s, under the extreme low temperature, living cellsmetabolism and growth activity is almost completely stopped, which is a kind of along effective method for plant germplasm preserved. G. straminea Maxim. andCodonopsis pilosula (Franch) Nannf. Are important medical plants in Gansu. G.macrophylla Maxim. is one of national key protected wild herbs which dormant budsis big and containing a high water content, C. pilosula (Franch) Nannf. is commonlyused in medicine which dormant buds is small and containing a low water content.The two types of dormant bud were systematically studied using vitrificationcryopreservation technology, which provided the technical foundation for widevariety of medicinal plants. Through the test the following conclusions were drawn:1. Dormant bud acted as explant of G. straminea Maxim., the best primarymedium was MS added with6-BA2.0mg/L, NAA0.01mg/L, sugar3%and agar0.7%; the best subculture medium was MS added with6-BA2.0mg/L and NAA0.01mg/L; the best rooting culture medium was1/2MS (half of mineral salts) addedwith IAA2mg/L, IBA0.5mg/L and sugar1.5%.2. Dormant bud acted as explant of C. pilosula (Franch.) Nannf., the best primarymedium was MS added with1.0mg/L6-BA and0.01mg/L NAA; the best subculturemedium was MS added with6-BA2.0mg/L and NAA0.01mg/L; the best rootingculture medium was1/2MS(half of macro elements) added with IAA2mg/L, IBA0.5mg/L, sugar1.5%and activated charcoal0.2%.3. A procedure of vitrification cryopreservation of dormant buds of G. stramineaMaxim. had been set up. Dormant buds took exercise of chilly winter withoutgerminating can be material of cryopreservation. Dormant buds shoot-tip,5-11mmlength, which was precultured on MS medium supplemented with0.7mol/L sucrose for3d, were loaded with2mol/L glycerol and0.4mol/L sucrose for40min at20℃,and then incunolated in PVS2for60min at20℃prior to a direct plunging into liquidnitrogen (LN). After rapid thawing in water at40℃for2min, the shoot-tips wererinsed in the MS medium supplemented with1.2mol/L sucrose for20min, and suckeddry the solution on the sterile filter paper, finally the shoot-tips were inoculation onMS medium supplemented with2.0mg/L6-BA, and0.01mg/L NAA for7d in darkprior to exposure to the light, the survival of shoot-tips was up to100%. Thetechnological system can be applied well in the same genus. The study found that theseasons taken dormant buds, the method of preculture, the dealing time of PVS2werethe key factors to cryopreservation. The dealing temperature of PVS2, unloading time,darkness culture time and the preservation period of LN had no obvious effects onpreservation results.4. A procedure of vitrification cryopreservation of dormant buds of C. pilosula(Franch.) Nannf. had been set up. The two-year old materials covered with roil werekept in4℃training for90days. The dormant buds with little root and bracts wereimmersed in2mol/L glycerol and0.4mol/L sucrose for20min at20℃, and thenincubated in PVS2for80min at20℃prior to a direct plunging into liquid nitrogen(LN). After rapid thawing in water at40℃for2min, the buds were rinsed in the MSmedium supplemented with1.2mol/L sucrose for20min, and sucked dry the solutionon the sterile filter paper, finally the buds were inoculation on MS mediumsupplemented with1.0mg/L6-BA, and0.05mg/L NAA, and cultured in the light,the survival of shoot-tips was up to74%. The state of dormant buds was critical, longtime of cooling experience can improve survival rate greatly. The cryopreservation ofthe whole dormant buds needs longer PVS2dealing time. The dormant buds withbracts a nd reco very c ulture unde r light were be ne fit for the ir s urviva l.5. In experiments of testing physiological and biochemical indexes duringvitrification cryopreservation of dormant buds of G. straminea Maxim., we found thatthe content of soluble saccharide and soluble protein increased by2.4and1.8timesrespectively at PVS2period, but the two indexes were remarkably decreased which islower than control after7d recovery culture. This illustrated that in order to answer the bad nature environment, the content of the two substances increased highly indormant buds. The dormants buds replied the adversity of vitrificationcryopreservation using the same methods. There is a high content of proline indormant buds, but during cryopreservation decreased suddenly. The content of starch,POD and SOD vigor had no obvious difference compared with the control whichindicated that the dormant buds had the ability of resisting adversity.6. There were some changes of ER in cells along with the treatments ofvitrification cryopreservation. The dormant buds had abundant smooth ER, in thecourse of vitrification cryopreservation, there was also rich smooth ER, but the cavityobviously swelled. In the course of recovery growth there was rough ER. Therefore,in the nature and artificial adversity, ER replied it with the same method. There weremany proplastid in control, but after loading and PVS2treatment, the proplastid beganto decrease along with appearance of starch grain, and the proplastid began to increasealong with the disappearing of starch grain after thawing. Dormant buds can replyadversity by mutual transformation. In the control there were a lot of vacuoles, afterpreculture the vacuoles became the more and smaller, and in the other period ofvitrification cryopreservation there were also a lot of vacuoles. This showed that theminiaturization of vacuoles may be the mechanism of answer the adversity of dormantbuds whatever in nature and artificial adversity, which was the reason the dormantbuds can obtain high survival rate after vitrification cryopreservation.7. An optimum reaction system of ISSR-PCR suitable for C. pilosula(Franch.)Nannf. was established, which can provide a standard procedure forevaluation of germplasm resources.25μL amplification reactions system containing30ng template DNA,2.5μL10×PCR Buffer, MgCl22.0mmol/L, dNTPs0.5mmol/L,primer0.4μmol/L and Taq DNA polymerase1.0U. Its reaction system is after apre-denaturing of5min at94℃,35cycles were performed with denaturing of30s at94℃, annealing of1min due to denaturing temperature of different primer, extensionof1.5min at72℃, a final extension step of7min at72℃and hold at4℃. The geneticstability of in vitro plantlets regenerated from cryopreserved dormant bud of C.pilosula (Franch.) Nannf. was tested, the results showed that the variation rate is only1.1%. 8. The genetic stability of in vitro plantlets regenerated from cryopreserveddormant bud of G. straminea Maxim. was tested using ISSR-PCR technology, theresults showed that no variation were found. |
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