Cell Culture And Cryopreservation Of Magnolia

Magnolia (Magnolia officinalis Rehd. Et Wils) is the Magnolia Magnolia (Magnoliaceae)(Magnolia) of woody plants. Magnolia whole body is treasure, is our country uses a variety ofeconomic trees, beverage, food, medicines, set wood and beautify the environment in one.Magnolia trees, beautiful tree, leaf, flower, big wide white pest and disease resistance, pollutionresistance, strong vitality, is today the streets, parks, residential district, garden city, gardengreening and landscaping ideal tree species. It with musk, licorice, Eucommia ulmoides wasclassified as a national mandatory management4important Chinese herbal medicines, are exportedto Southeast Asia and japan. Magnolia bark extract is rich in chemicals, according to our countrythe earliest pharmaceutical monograph" Shennong materia medica by" records, Magnolia isaromatized dampness. Magnolia officinal Magnolia has a long history, is widely distributed, but isa primitive type, for the study of East Asia and North America flora and Magnolia classificationhas scientific significance.However, long-term since suffer natural disasters and human on wild medicinal plantresources in the predatory exploitation, makes a lot of the wild resources of medicinal plants areon the verge of exhaustion, With the growth of the population and the resources of medicinalplants of the sharp increase in demand, resulting in a" feral resource decreases ceaselessly, hasbeen listed as the second class of the national protected wild plants. On the verge of exhaustion ofmagnolol in germplasm resources conservation has become the crucial question.This study use of plant cell culture technology, The Magnolia officinalis callus tissue cultureand cell suspension culture system on the, Through the study of different factors on suspensioncell growth effects of Magnolia officinalis, the establishment of a rapid growth of cell suspensionculture system, For the use of cell culture technology to produce the effective chemicalconstituents in bark to provide some reference. For the Magnolia officinalis resource regenerationand germplasm preservation provided valuable technical information and important theoreticalbasis, but on its suspension cell culture cryopreservation research does not see more. Therefore,the test is based on previous researches, to be improved and perfected, the Magnolia suspensionculture cell cryopreservation of germplasm resources preservation measures, as Magnoliaprovided experimental basis. The main results are as follows:1.Callus induction and subculture: in Cortex Magnoliae Officinalis callus inductionexperiment, various medium large range can be generated by callus, but the test results show that,with B₅NAA1.0mg L^-16-BA1.0mg L^-1 as the most suitable medium; B₅＋2,4-D2.0mg·L^-1＋6-BA1.0mg·L^-1medium most conducive to callus differentiation. The best of apical buds and stem segment explants is; additional concentration of3.00mg·L^-1ascorbic acid most conducive toprevent Callus Browning; different hormones on callus induction effect of Magnolia officinalis are2,4-D>6-BA> NAA; callus culture of35d as the best harvest time.2.Suspension cell culture system: in Cortex Magnoliae officinalis in cell suspension culture,studied the basic culture medium, inoculation quantity, hormone combinations, sucroseconcentration, shaker speed, pH, aeration on the growth of suspension culture cells influence.Magnolia cell suspension culture system is optimized, the test results show that, the optimalMagnolia cell growth medium is B₅＋NAA1.0mg·L^-1＋6-BA1.0mg·L^-1and B₅＋2,4-D2.0mg·L^-1＋6-BA1.0mg·L^-1; optimal inoculum for0.1g/mL; sucrose concentration was30g·L^-1is mostbeneficial to the cell growth; optimum rpm110rpm; Magnolia cell suspension culture and liquidculture medium pH value in5.6～5.8between the most suitable cell growth of Magnoliaofficinalis; cell suspension culture of cotton plug sealing effect is best; cell culture cycle for24d.3.Magnolia suspension cell cryopreservation research:the Magnolia suspension cellcryopreservation of several main factors of single factor experiment, research and analysis ofMagnolia officinalis suspension cell physiological state and all kinds of frozen pretreatment oncell viability after cryopreservation effect. The test results show that, after several times of preculture subculture of6d, in a vigorous growth period, the cell using fractional freezing,cryopreservation of life power;"10%DMSO＋5%Suc " as a cryoprotectant on cryopreservation ofsuspension cells of Cortex Magnoliae Officinalis plays the best protection effect, is the idealchoice of cryoprotectant; better cooling procedure is Magnolia cell suspension into the freezingpipe, at0℃ice water pretreatment with30min, and then put into the refrigerator(一80℃), with0.5℃/rain cooling speed from0℃to30℃, at this temperature for1h, then plunged into liquidnitrogen (一196℃). Suspension cells in liquid nitrogen preservation of10d, out with a30℃water thawing effect is better; for cryopreservation of cells after restorative growth for validation,found in dark environment conducive to cell recovery growth.To sum up, study of Magnolia officinalis cell culture and cryopreservation, to promote theactive pharmaceutical ingredients and germplasm preservation technology research has positivesignificance.