CN105638645A - Method for carrying out encapsulation-vitrification cryopreservation on suspension cells of taxus chinensis - Google Patents

Abstract

The invention discloses a method for embedment and vitrification ultra-low temperature preservation of yew suspension cells. Including: (1) Suspension culture of Taxus chinensis callus: pick white or light yellow callus cultured for 15 days, use the improved MS-I medium; (2) Taxus chinensis cell line suspension pre-culture: use The modified MS-II medium was pre-cultured for 1 day; (3) Embedding; (4) Pretreatment: Treat the rubber balls with pre-loading solution LB7 for 30-60 minutes; (5) Loading: Treat the rubber balls with PVS3 30-60 minutes; (6) cryopreservation; (7) thawing: use a 45°C constant temperature water bath to thaw for about 1 minute; (8) restore the culture, and the growth can be resumed in about 6 weeks. The method of the invention is simple and easy to operate, has short restoration growth time, is suitable for the preservation of large-scale yew germplasm resources, is suitable for the preservation of various yew cell lines, and has wide-spectrum adaptability.

Description

A method for ultra-low temperature preservation of yew suspension cells embedded in vitrification

technical field

The invention relates to a method for ultra-low temperature preservation of yew suspended cells embedded in vitrification, and belongs to the technical field of yew seed resource preservation.

Background technique

Taxus (Taxus) Taxaceae (Taxaceae) evergreen trees, mainly distributed in my country are Chinese yew (Taxuschinensis), southern yew (T.chinensisvar.mairei), northeast yew (T.cuspidata), Yunnan red bean Chinese fir (T.yunnanensis) and Tibetan yew (T.wallichinana) (Zhang Zongqin, Liu Zhiming. Taxus. Northwest A&F University Press, 2010, 10:1). Taxus plants contain active ingredients such as diterpenoids, triterpenes, steroids, high terpenoids, flavonoids, phenylpropanoids, etc. Among them, there are more than 560 kinds of terpenoids (Zu Yuangang. Southern Red Bean High-efficiency processing and utilization technology of Chinese fir renewable resources. Science Press, 2010.2: 13), the most famous is that paclitaxel in isopentenyl diterpene has a significant effect in the treatment of cancer, and Taxol is a registered commercial medical patent drug of BMS company in the United States ( Sonia Malike tal. Production of the anticancer drug taxolin Taxus baccata suspension cultures:A Review. Process Biochemistry, 2011(46):23-34). The use of paclitaxel in the treatment of cancer began in 1979 when it was discovered that it caused cells to stagnate in the G2/M phase during cell mitosis (SchiffPB, HorwitzSB. Taxiolstabilizesmicrotubules in mouse fibroblast cells. ProcNatlAcadSciUSA, 1980, 77: 1S61-65), and has been widely used in breast cancer , lung cancer, small cell carcinoma, etc., the market price of paclitaxel once reached 600,000 US dollars per kilogram (Smetanskal. Production of secondary metabolites using plant cell cultures. Food Biotechnol, 2008, 111: 187-228). As a successful anticancer drug, the annual market value of paclitaxel has exceeded 100 million US dollars (MalikS, et al. Production of the anticancer drug taxolin Taxus baccata suspension cultures: Areview. Process Biochemistry, 2011 (46): 23-34). Being plundered, it is on the verge of extinction. In August 1999, my country listed yew as a national first-class endangered plant (Shi Qingwen, et al. Chemical Research on Taxus Plants-Research on Taxane Compounds. Chemical Industry Press, 2013, 1:1).

Paclitaxel was initially extracted from the bark, but its content was extremely low, and the growth of Taxus plants was extremely slow, and the extraction process was complicated and the environment was damaged. Later, chemical synthesis method, semi-synthesis method, heterologous expression system and cell suspension culture were developed. Cell suspension culture can produce target compounds continuously and consistently because of its biological yield is not limited by geographical location and season. It has the advantages of being converted into renewable resources and being environmentally friendly, and it is very easy to realize factory production (Susan Howat, et al. Paclitaxel: biosynthesis, production and future prospects. New Biotechnology, 2014, 3(31): 242-245). Taxus cell suspension culture is also limited by factors such as genetic instability, heterogeneity in the culture process, slow growth relative to fungal culture, and unstable content of the target product in the large-scale fermentation culture process (KoleweME, et al. Pharmaceutically active natural product synthesis and supply via plant cell culture technology. MolPharm, 2008, 5: 243-256). Therefore, it is one of the important links of taxus cell suspension culture to screen high-yield paclitaxel cell lines and reduce multiple subcultures of core germplasm resources to reduce genetic variation and pollution.

The preservation of plant germplasm resources mainly includes methods such as in situ preservation of populations, outdoor in vitro preservation, and in vitro culture and slow growth preservation. Different plant species and preservation material types have different preservation methods. Among all conservation methods, cryopreservation can be applied to the conservation of biological germplasm resources relying on vegetative reproduction methods. Different plant cells, tissues and organs including suspension cells, pollen, embryoid body cultures, somatic embryos, zygotic embryos and shoot tips can be used as explants for cryopreservation (ME González-Benito, et al. Review. The use of cryopreservation for germ plasma conservation of vegetatively propagated crops .Spanish Journal of Agriculture Research, 2004, 2(3):341-351). Most living cells contain high water content and are extremely sensitive to low temperatures of 0°C. Using cryoprotectants combined with slow cooling methods can preserve larger structures such as shoot tip materials. In recent decades, the vitrification cryopreservation method, the embedding dehydration and drying cryopreservation method, and the droplet preservation method have been newly developed. KimSI and others have successfully reported the method of cryopreservation of Taxus chinensis in suspension culture for 7 days by using the method of programmed cooling (KimSI, et al.CryopreservationofTaxuschinensissuspensioncellcultures.Cryoletters, 2000,22(1):43-50). Ye Fang et al. used the method of programmed cooling to study the ultra-low temperature preservation method of Taxus chinensis, and obtained a relative value of TTC activity of Taxus chinensis suspension cells of 4.33% (Ye Fang, et al. Research on factors related to ultra-low temperature preservation of Taxus chinensis callus. Wuhan Botany Research, 2001, 19(4): 327-331). Zang Xin et al. adopted the method of programmed cooling for the suspension culture of Taxus chinensis cells for 16 days, and successfully verified the restorative growth of cells (Zang Xin, et al. Cryogenic preservation of Taxus chinensis suspension culture cells. Biotechnology, 2002, 2(12): 13-14). K et al. used a simplified program-controlled cooling box combined with cryopreservation to preserve the cell suspension lines of Taxus × media Rehd and Taxus floridana Nutt. The survival rate of suspension cells has a significant relationship with the cell line ( K, et al. Cryopreservation of cells suspension cultures of Taxus×media and Taxusfloridana. Biologia Plantarum, 2008, 52(2):329-333). Wu Wenjuan established the cryopreservation method of vitrification method, pre-culture drying cryopreservation method and desiccation/vitrification cryopreservation method of Taxus chinensis and Taxus Mandia, but the survival rate of cell suspension was low (Wu Wenjuan. Taxus cell line Germplasm conservation and genetic variation research. Huazhong University of Science and Technology, 2009). Existing studies have significant deficiencies, such as the need to use expensive program-controlled cooling devices or other similar equipment, cell genotype dependence, and low survival rates.

Embedded vitrification cryopreservation method is a kind of cryopreservation method that first embeds materials with a curing agent and then combines vitrification. It does not require any physical drying and dehydration process. It has been widely used in the isolation of plant stem tips and stem segments体超低温保存(M.E.González-Benito,etal.Review.Theuseofcryopreservationforgermplasmconservationofvegetativelypropagatedcrops.SpanishJournalofAgricultureResearch,2004,2(3):341-351；JeonSM,etal.Applicationofencapsulation-vitrificationincombinationwithairdehydrationenhancescryotoleranceofChrysanthemummorifoliumshootstips.ScientiaHorticulturae,2015,194:91-99；TahtamouniR,etal. In Vitro Conservation and Cryopreservation of Medicinal and Aromatic Plants: A Review. 2015). So far there is no report on the ultra-low preservation of yew suspension cells based on this method.

Contents of the invention

The purpose of the present invention is to provide a method for embedment and vitrification cryogenic preservation of suspension cells of yew plants, in order to solve the problems of continuous subculture of core germplasm in the suspension culture process of yew cell lines, variation and pollution in the preservation process, etc. , can be applied to the long-term preservation of yew cell suspension line cell resources and the construction process of yew plant seed resource bank.

The purpose of the present invention is achieved in the following ways.

A kind of yew suspension cell embedding vitrification cryopreservation method, comprises the following steps:

(1) Suspension culture of yew callus: pick yew callus and inoculate it in the improved MS-I liquid medium for suspension culture, the improved MS-I liquid medium is: 2500mg/LKNO ₃ , 2500mg /LNH ₄ NO ₃ , 170mg/LKH ₂ PO ₄ , 370mg/LMgSO ₄ 7H ₂ O, 440mg/LCaCl ₂ 2H ₂ O, 37.3mg/LNa ₂ -EDTA, 27.8mg/LFeSO ₄ 7H ₂ O, 100mg /L Inositol, 0.5mg/L Niacin, 0.5mg/L Pyridoxine Hydrochloride, 0.1mg/L Thiamine Hydrochloride, 2mg/L Glycine, 0.83mg/LKI, 6.2mg/LH ₃ BO ₃ , 22.3mg /LMnSO ₄ 4H ₂ O, 8.6mg/LZnSO ₄ 7H ₂ O, 0.25mg/L, Na ₂ MoO ₄ 2H ₂ O, 0.025mg/LCuSO ₄ 5H ₂ O, 0.025mg/LCoCl ₂ 6H ₂ O, 30g/L sucrose, 1~3mg/Lpicloram, 0.5~1mg/L6-BA, dilute to 1L with distilled water, pH 5.8;

(2) Taxus cell line suspension pre-cultivation: Get the well-dispersed Taxus suspension cells obtained in step (1) and inoculate them in the improved MS-II liquid medium for suspension culture; the improved MS-II liquid medium is: 1000mg /LKNO ₃ , 500mg/LKCl, 800mg/LNH ₄ NO ₃ , 170mg/LKH ₂ PO ₄ , 370mg/LMgSO ₄ 7H ₂ O, 440mg/LCaCl ₂ 2H ₂ O, 37.3mg/LNa ₂ -EDTA, 27.8mg /LFeSO ₄ 7H ₂ O, 100mg/L inositol, 0.5mg/L niacin, 0.5mg/L pyridoxine hydrochloride, 0.1mg/L thiamine hydrochloride, 2mg/L glycine, 0.83mg/LKI, 6.2 mg/LH ₃ BO ₃ , 22.3mg/LMnSO ₄ 4H ₂ O, 8.6mg/LZnSO ₄ 7H ₂ O, 0.25mg/L, Na ₂ MoO ₄ 2H ₂ O, 0.025mg/LCuSO ₄ 5H ₂ O , 0.025mg/LCoCl ₂ 6H ₂ O, 30g/L sucrose, 60g/L LD-sorbitol, 1~3mg/Lpicloram, 0.5~1mg/L6-BA, dilute to 1L with distilled water, pH 5.8;

(3) Embedding: the yew cells in step (2) are mixed with sodium alginate embedding solution, and dropped into the Ca2 ⁺ solution to form glue balls containing yew cells;

(4) Pretreatment: the immobilized rubber balls containing Taxus cells obtained in step (3) are pretreated in the preloading solution;

(5) Loading: take the rubber balls after the pretreatment in step (4) and use the loading liquid to carry out vitrification treatment;

(6) Cryopreservation: take the rubber balls after vitrification treatment in step (5), replace them with the same vitrification solution stored at 4°C, and put them together with the cryopreservation tubes in liquid nitrogen for storage immediately.

The yew callus in the step (1) is the white or light yellow callus of Taxus chinensis, or the white or light yellow callus of Taxus chinensis, or the white or light yellow callus of Taxus yunnanensis, or Mandia yew white or yellowish callus.

Step (1) Pick the Taxus chinensis callus after 15 days of subculture on the solid medium for inoculation, and carry out suspension culture in a constant temperature shaker at 100-120 rpm under the condition of 22-25 ° C, the callus inoculum amount It is 5-10% of the weight of the medium; it is subcultured once every 7-10 days; it is used for inoculation in step (2) after 15-20 days of primary culture.

In the step (2), the well-dispersed yew suspension cells obtained in the step (1) are inoculated in the improved MS-II liquid medium for suspension culture for 1 day, and the conditions are 22-25 ° C, 100-120 rpm constant temperature Shaking bed culture, the inoculum amount is 5-10% of the weight of the culture medium after the cells cultured in the suction filtration step (1).

Step (3) Suction filter and weigh the same mass of yew cells cultured in step (2) and mix with sodium alginate embedding solution, drop into 0.1 mol/L Ca ²⁺ solution with a sterile pipette tip to form a yew cell containing yew Glue balls of cells.

The formula of sodium alginate embedding solution in step (3) is: 3g/L sodium alginate, 180g/L glycerin, 1000mg/LKNO ₃ , 500mg/LKCl, 800mg/LNH ₄ NO ₃ , 170mg/LKH ₂ PO ₄ , 370mg /LMgSO ₄ 7H ₂ O, 37.3mg/LNa ₂ -EDTA, 27.8mg/LFeSO ₄ 7H ₂ O, 100mg/L inositol, 0.5mg/L niacin, 0.5mg/L pyridoxine hydrochloride, 0.1mg /L Thiamine Hydrochloride, 2mg/L Glycine, 0.83mg/LKI, 6.2mg/LH ₃ BO ₃ , 22.3mg/LMnSO ₄ 4H ₂ O, 8.6mg/LZnSO ₄ 7H ₂ O, 0.25mg/LNa ₂ MoO ₄ 2H ₂ O, 0.025mg/LCuSO ₄ 5H ₂ O, 0.025mg/LCoCl ₂ 6H ₂ O, 30g/L sucrose, 1～3mg/Lpicloram, 0.5～1mg/L6-BA, dilute to volume with distilled water to 1L with a pH of 5.8.

The preloading liquid in step (4) is LB7, and the formula is: 300g/L glycerin, 150g/L dimethyl sulfoxide, 400g/L sucrose, 150g/L trehalose, 2500mg/LKNO ₃ , 2500mg/LNH ₄ NO ₃ , 170mg/LKH ₂ PO ₄ , 370mg/LMgSO ₄ 7H ₂ O, 440mg/LCaCl ₂ 2H ₂ O, 37.3mg/LNa ₂ -EDTA, 27.8mg/LFeSO ₄ 7H ₂ O, 100mg/L inositol , 0.5mg/L niacin, 0.5mg/L pyridoxine hydrochloride, 0.1mg/L thiamine hydrochloride, 2mg/L glycine, 0.83mg/LKI, 6.2mg/LH ₃ BO ₃ , 22.3mg/LMnSO ₄ · 4H ₂ O, 8.6mg/LZnSO ₄ 7H ₂ O, 0.25mg/LNa ₂ MoO ₄ 2H ₂ O, 0.025mg/LCuSO ₄ 5H ₂ O, 0.025mg/LCoCl ₂ 6H ₂ O, 1～3mg/ Lpicloram, 0.5~1mg/L6-BA, dilute to 1L with distilled water; the preloading time is 30~60 minutes. Put about 50 glue balls of yew cells per 100 ml of preloading solution.

In step (5), the loading solution PVS3 is used for vitrification for 30-60 minutes; PVS3 uses liquid MS basic medium as a solute, which contains 40% by mass of glycerol and 40% by mass of sucrose. Take 50 pretreated glue balls and mix them with 100 ml of loading solution.

The process of thawing and restoring culture after the above method is as follows:

Thawing: Take the rubber balls in step (6) and quickly put them into 45°C waters and shake the cryopreservation tube continuously, and the thawing is completed in 1 minute;

Recovery culture: Remove the vitrification solution of the thawed glue beads, soak the glue beads in liquid MS medium for 10 minutes, repeat twice, inoculate on MS solid medium for dark culture, and subculture once every 15 days. 22±1°C; continue dark culture for at least 6 weeks, the resurrection rate of yew gumballs can reach 91.11%.

The present invention has the following advantages: (1) the present invention has determined the preservation method specially aimed at the yew cell suspension system through the exploration of the culture conditions of each step, and finds that it can be applied to southern yew, northeast yew, and Yunnan yew Compared with previous studies, the cell suspension system of Taxus mandia has broad-spectrum adaptability; (2) the required equipment of the present invention is simple, does not require a program-controlled cooling device or similar equipment, and is simple and easy to operate; (3) The present invention is suitable for the preservation of large-scale yew germplasm resources, and the culture can be resumed after cryopreservation, and the growth can be resumed within 6 weeks, and the suspension culture can be carried out after a large amount of multiplication.

Description of drawings

Fig. 1 is that the yew suspension cell embedding in the present invention restores the culture growth situation;

Wherein A. Taxus chinensis suspension cells were embedded without subsequent treatment and the cells were recovered and cultured for 4 weeks in glue balls;

B. After the suspension cells of Taxus chinensis were embedded, the cells were recovered and cultured for 8 weeks without subsequent treatment;

C. The yew suspension cells were preserved at low temperature to restore the rubber balls cultured for 4 weeks;

D. Suspension cells of Taxus chinensis were cryopreserved to restore the glue balls cultured for 8 weeks.

detailed description

The following examples are intended to further illustrate the present invention without limiting it.

The present invention has done a lot of screening for the culture conditions of each step, as well as the medium formula, etc., and the following are some screening tests and results.

Step (2) Taxus cell line suspension pre-culture time screening of the present invention

Pre-cultivation time (days) Yew gum ball revival rate (%) 0 33.42 1 91.11 3 12.04 5 0 7 0

The present invention step (4) the screening of yew gum ball preloading liquid kind

Composition of preloading fluid Yew gum ball revival rate (%) 2M glycerol + 0.4M sucrose 0 2M glycerol + 0.6M sucrose 1.88 2M glycerol + 0.8M sucrose 20.36 2M glycerin + 1.0M sucrose 10.57 30%LB7 0 60%LB7 30.25 80%LB7 61.41 100% LB7 91.11

The screening of step (4) yew gum ball LB7 pretreatment time of the present invention

Preprocessing time (minutes) Yew gum ball revival rate (%) 0 26.01 30 89.12 60 91.11 90 70.25 120 62.12 180 42.36

Screening of step (5) yew gum ball PSV3 vitrification treatment time of the present invention

Example 1: Southern Taxus cell line 1b, preserved for this experiment

A method for ultra-low temperature preservation of yew suspension cell lines by embedding vitrification method, the steps are as follows:

(1) Pick the white or light yellow yew callus that has been subcultured on the solid for 15 days and inoculate it in the improved MS-I liquid culture for suspension culture, the conditions are 22-25 ° C, 100-120 rpm Constant temperature shaker culture, callus inoculation amount is 5-10% of medium weight; Suspension culture is subcultured once every 10 days, and used for step (2) inoculation after primary culture for 15-20 days. MS-I medium formula is as follows:

2500mg/LKNO ₃ , 2500mg/LNH ₄ NO ₃ , 170mg/LKH ₂ PO ₄ , 370mg/LMgSO ₄ 7H ₂ O, 440mg/LCaCl ₂ 2H ₂ O, 37.3mg/LNa ₂ -EDTA, 27.8mg/LFeSO ₄ 7H ₂ O, 100mg/L inositol, 0.5mg/L niacin, 0.5mg/L pyridoxine hydrochloride, 0.1mg/L thiamine hydrochloride, 2mg/L glycine, 0.83mg/LKI, 6.2mg/LH 3BO ₃ , 22.3mg/LMnSO ₄ 4H ₂ O, 8.6mg/LZnSO ₄ 7H ₂ O, 0.25mg/L, Na ₂ MoO ₄ 2H ₂ O, 0.025mg/ _{LCuSO 4 5H 2} O, 0.025mg /LCoCl ₂ ·6H ₂ O, 30g/L sucrose, 1~3mg/Lpicloram, 0.5~1mg/L6-BA, dilute to 1L with distilled water, pH value is 5.8.

(2) Take the well-dispersed Taxus chinensis suspension cells or cell microaggregates by suction filtration, inoculate them on MS-II medium and pre-culture for 1 day, the conditions are 22-25°C, 100-120 rpm constant temperature shaker culture, The inoculum amount is 5-10% of the weight of the culture medium after the cultured cells in the suction filtration step (1).

The formula of MS-II medium is as follows:

_{27.8 _ _ _ _ _ _ _ _ _} mg/LFeSO ₄ ·7H ₂ O, 100mg/L inositol, 0.5mg/L niacin, 0.5mg/L pyridoxine hydrochloride, 0.1mg/L thiamine hydrochloride, 2mg/L glycine, 0.83mg/LKI, 6.2mg/LH ₃ BO ₃ , 22.3mg/LMnSO ₄ 4H ₂ O, 8.6mg/LZnSO ₄ 7H ₂ O, 0.25mg/L, Na ₂ MoO ₄ 2H ₂ O, 0.025mg/LCuSO ₄ 5H ₂ O, 0.025mg/LCoCl ₂ 6H ₂ O, 30g/L sucrose, 60g/L LD-sorbitol, 1~3mg/Lpicloram, 0.5~1mg/L6-BA, dilute to 1L with distilled water, the MS culture medium The pH is 5.8.

(3) Suction filter and weigh the same mass of yew cells pre-cultured for 1 day, mix with sodium alginate embedding solution, drop into the Ca ²⁺ solution (0.1MCaCl ₂ solution) with a sterile pipette tip, and wait for about 1 minute Form a 0.4 cm rubber ball containing yew cells; the embedding solution formula is: 3g/L sodium alginate, 180g/L glycerol, 1000mg/LKNO ₃ , 500mg/LKCl, 800mg/LNH ₄ NO ₃ , 170mg/LKH ₂ PO ₄ , 370mg/LMgSO ₄ 7H ₂ O, 37.3mg/LNa ₂ -EDTA, 27.8mg/LFeSO ₄ 7H ₂ O, 100mg/L inositol, 0.5mg/L niacin, 0.5mg/L pyrimidine hydrochloride Pyridoxine, 0.1mg/L Thiamine Hydrochloride, 2mg/L Glycine, 0.83mg/LKI, 6.2mg/LH ₃ BO ₃ , 22.3mg/LMnSO ₄ 4H ₂ O, 8.6mg/LZnSO ₄ 7H ₂ O, 0.25mg/L, Na ₂ MoO ₄ 2H ₂ O, 0.025mg/LCuSO ₄ 5H ₂ O, 0.025mg/LCoCl ₂ 6H ₂ O, 30g/L sucrose, 1～3mg/Lpicloram, 0.5～1mg/L6 -BA, make up to 1 L with distilled water, pH 5.8.

(4) Pretreat the immobilized rubber balls in preloading solution LB7 for 30 minutes. L trehalose, 2500mg/LKNO ₃ , 2500mg/LNH ₄ NO ₃ , 170mg/LKH ₂ PO ₄ , 370mg/LMgSO ₄ 7H ₂ O, 440mg/LCaCl ₂ 2H ₂ O, 37.3mg/LNa ₂ -EDTA, 27.8 mg/LFeSO ₄ ·7H ₂ O, 100mg/L inositol, 0.5mg/L niacin, 0.5mg/L pyridoxine hydrochloride, 0.1mg/L thiamine hydrochloride, 2mg/L glycine, 0.83mg/LKI, 6.2mg/LH ₃ BO ₃ , 22.3mg/LMnSO ₄ 4H ₂ O, 8.6mg/LZnSO ₄ 7H ₂ O, 0.25mg/L, Na ₂ MoO ₄ 2H ₂ O, 0.025mg/LCuSO ₄ 5H ₂ O, 0.025mg/LCoCl ₂ ·6H ₂ O, 1~3mg/Lpicloram, 0.5~1mg/L6-BA, dilute to 1L with distilled water. Put about 50 glue balls of yew cells per 100 ml of preloading solution.

(5) Vitrify the preloaded rubber balls for 30 minutes using the loading solution PVS3 vitrification solution. The formulation of PVS3 is 40wt% glycerol (using liquid MS basic medium as solute) mixed with 40wt% sucrose (using liquid MS The minimal medium is the solute). Mix 50 pretreated gumballs with 100 ml of loading solution

(6) Take the loaded cryopreservation tubes stored at 4°C, fill each tube with 8 beads, and then fill the 2.5ml cryopreservation tubes with PVS3 vitrification solution stored at 4°C, close the cap tightly and put them away immediately. Stored in liquid nitrogen; after being put into liquid nitrogen, it can be stored permanently, and can be restored and cultivated when necessary.

(7) Take out the preserved cryopreservation tube with rubber beads from the liquid nitrogen, immediately put it into a 45°C constant temperature water pot, shake the cryopreservation tube constantly, wait for about 1 minute for the ice crystals to melt, and insert the cryopreservation tube into the ice Reserve in the bath.

(8) Take the rubber balls out of the cryopreservation tube, put the MS culture into the liquid and soak the rubber beads at room temperature for about 10 minutes, replace the fresh MS medium and repeat the operation; then inoculate the rubber beads into the MS medium Culture in the dark at 22±1°C, and subculture once every 15 days; the culture temperature is 22±1°C; after 6 weeks of continuous dark culture, the revival rate of yew gumballs can reach 91.11%. Subculture medium formula: 1900mg/LKNO ₃ , 1650mg/LNH ₄ NO ₃ , 170mg/LKH ₂ PO ₄ , 370mg/LMgSO ₄ 7H ₂ O, 440mg/LCaCl ₂ 2H ₂ O, 37.3mg/LNa ₂ -EDTA, 27.8mg/LFeSO ₄ 7H ₂ O, 100mg/L inositol, 0.5mg/L niacin, 0.5mg/L pyridoxine hydrochloride, 0.1mg/L thiamine hydrochloride, 2mg/L glycine, 0.83mg/LKI, 6.2mg/LH ₃ BO ₃ , 22.3mg/LMnSO ₄ 4H ₂ O, 8.6mg/LZnSO ₄ 7H ₂ O, 0.25mg/L, Na ₂ MoO ₄ 2H ₂ O, 0.025mg/L LCuSO ₄ ·5H ₂ O, 0.025mg/LCoCl ₂ ·6H ₂ O, 30g/L sucrose, 1~3mg/Lpicloram, 0.5~1mg/L6-BA, 6g/L agar, dilute to 1L with distilled water, the The pH of the medium was 5.8.

Example 2

In this example, the immobilized rubber balls were pretreated in preloading solution LB7 for 60 minutes. The formula for LB7 preloading was: 300g/L glycerin, 150g/L dimethyl sulfoxide, 400g/L sucrose, 150g /L Trehalose, 2500mg/LKNO ₃ , 2500mg/LNH ₄ NO ₃ , 170mg/LKH ₂ PO ₄ , 370mg/LMgSO ₄ 7H ₂ O, 440mg/LCaCl ₂ 2H ₂ O, 37.3mg/LNa ₂ -EDTA, 27.8mg/LFeSO ₄ 7H ₂ O, 100mg/L inositol, 0.5mg/L niacin, 0.5mg/L pyridoxine hydrochloride, 0.1mg/L thiamine hydrochloride, 2mg/L glycine, 0.83mg/LKI , 6.2mg/LH ₃ BO ₃ , 22.3mg/LMnSO ₄ 4H ₂ O, 8.6mg/LZnSO ₄ 7H ₂ O, 0.25mg/L, Na ₂ MoO ₄ 2H ₂ O, 0.025mg/LCuSO ₄ 5H ₂ O, 0.025 mg/LCoCl ₂ ·6H ₂ O, 1-3 mg/L picloram, 0.5-1 mg/L 6-BA, dilute to 1 L with distilled water;

Example 3

In this embodiment, the preloaded rubber balls were vitrified for 60 minutes using the loading liquid PVS3 vitrification solution. The formulation of PVS3 was 40wt% glycerol (using liquid MS basic medium as solute) mixed with 40wt% sucrose (using liquid MS basal medium is a solute); all the other implementation steps are the same as in Example 1.

Example 4

In this example, the white or yellowish Taxus chinensis callus that was subcultured in solid for 15 days was picked and inoculated in the improved MS-I liquid culture for suspension culture, and the suspension culture was subcultured once every 10 days, MS -No. I culture medium formula is as follows:

2500mg/LKNO ₃ , 2500mg/LNH ₄ NO ₃ , 170mg/LKH ₂ PO ₄ , 370mg/LMgSO ₄ 7H ₂ O, 440mg/LCaCl ₂ 2H ₂ O, 37.3mg/LNa ₂ -EDTA, 27.8mg/LFeSO ₄ 7H ₂ O, 100mg/L inositol, 0.5mg/L niacin, 0.5mg/L pyridoxine hydrochloride, 0.1mg/L thiamine hydrochloride, 2mg/L glycine, 0.83mg/LKI, 6.2mg/LH 3BO ₃ , 22.3mg/LMnSO ₄ 4H ₂ O, 8.6mg/LZnSO ₄ 7H ₂ O, 0.25mg/L, Na ₂ MoO ₄ 2H ₂ O, 0.025mg/ _{LCuSO 4 5H 2} O, 0.025mg /LCoCl ₂ ·6H ₂ O, 30g/L sucrose, 1-3mg/Lpicloram, 0.5-1mg/L6-BA, distilled water to 1L, pH value 5.8;

Example 5

In this example, the white or light yellow Taxus yunnanensis callus that was subcultured on the solid for 15 days was picked and inoculated in the improved MS-I liquid culture for suspension culture, and the suspension culture was subcultured once every 10 days, MS -No. I culture medium formula is as follows:

2500mg/LKNO ₃ , 2500mg/LNH ₄ NO ₃ , 170mg/LKH ₂ PO ₄ , 370mg/LMgSO ₄ 7H ₂ O, 440mg/LCaCl ₂ 2H ₂ O, 37.3mg/LNa ₂ -EDTA, 27.8mg/LFeSO ₄ 7H ₂ O, 100mg/L inositol, 0.5mg/L niacin, 0.5mg/L pyridoxine hydrochloride, 0.1mg/L thiamine hydrochloride, 2mg/L glycine, 0.83mg/LKI, 6.2mg/LH 3BO ₃ , 22.3mg/LMnSO ₄ 4H ₂ O, 8.6mg/LZnSO ₄ 7H ₂ O, 0.25mg/L, Na ₂ MoO ₄ 2H ₂ O, 0.025mg/ _{LCuSO 4 5H 2} O, 0.025mg /LCoCl ₂ 6H ₂ O, 30g/L sucrose, 1~3mg/Lpicloram, 0.5~1mg/L6-BA, distilled water to 1L, the pH of the MS culture solution is 5.8; the rest of the implementation steps are the same example 1.

Example 6

In this embodiment, the white or light yellow Taxus mandia callus that was subcultured on the solid for 15 days was picked and inoculated in the improved MS-I liquid culture for suspension culture, and the suspension culture was subcultured once every 10 days , No. MS-I culture medium formula is as follows:

2500mg/LKNO ₃ , 2500mg/LNH ₄ NO ₃ , 170mg/LKH ₂ PO ₄ , 370mg/LMgSO ₄ 7H ₂ O, 440mg/LCaCl ₂ 2H ₂ O, 37.3mg/LNa ₂ -EDTA, 27.8mg/LFeSO ₄ 7H ₂ O, 100mg/L inositol, 0.5mg/L niacin, 0.5mg/L pyridoxine hydrochloride, 0.1mg/L thiamine hydrochloride, 2mg/L glycine, 0.83mg/LKI, 6.2mg/LH 3BO ₃ , 22.3mg/LMnSO ₄ 4H ₂ O, 8.6mg/LZnSO ₄ 7H ₂ O, 0.25mg/L, Na ₂ MoO ₄ 2H ₂ O, 0.025mg/ _{LCuSO 4 5H 2} O, 0.025mg /LCoCl ₂ 6H ₂ O, 30g/L sucrose, 1~3mg/Lpicloram, 0.5~1mg/L6-BA, distilled water to 1L, the pH of the MS culture solution is 5.8; the rest of the implementation steps are the same example 1.

The revival rate of the yew gum balls in Examples 1-6 of the present invention can reach more than 88%, and the highest can reach 91.11%.

Claims (9)

1. a Ramulus et folium taxi cuspidatae suspension cell encapsulation-vitrification cryopreservation method, it is characterised in that comprise the following steps:

(1) suspension culture of taxus callus: picking taxus callus is inoculated in the MS-I fluid medium of improvement and carries out suspension culture, the MS-I fluid medium of improvement is: 2500mg/LKNO₃, 2500mg/LNH₄NO₃, 170mg/LKH₂PO₄, 370mg/LMgSO₄��7H₂O, 440mg/LCaCl₂��2H₂O, 37.3mg/LNa₂-EDTA, 27.8mg/LFeSO₄��7H₂O, 100mg/L inositol, 0.5mg/L nicotinic acid, 0.5mg/L pyridoxine hydrochloride, 0.1mg/L thiamine hydrochloride, 2mg/L glycine, 0.83mg/LKI, 6.2mg/LH₃BO₃, 22.3mg/LMnSO₄��4H₂O, 8.6mg/LZnSO₄��7H₂O, 0.25mg/L, Na₂MoO₄��2H₂O, 0.025mg/LCuSO₄��5H₂O, 0.025mg/LCoCl₂��6H₂O, 30g/L sucrose, 1��3mg/Lpicloram, 0.5��1mg/L6-BA, with distilled water constant volume to 1L, pH value is 5.8; (2) taxus chinensis clone suspension preculture: take the finely disseminated Ramulus et folium taxi cuspidatae suspension cell obtained in step (1) and be inoculated in suspension culture in the MS-II fluid medium of improvement; Improvement MS-II fluid medium is: 1000mg/LKNO₃, 500mg/LKCl, 800mg/LNH₄NO₃, 170mg/LKH₂PO₄, 370mg/LMgSO₄��7H₂O, 440mg/LCaCl₂��2H₂O, 37.3mg/LNa₂-EDTA, 27.8mg/LFeSO₄��7H₂O, 100mg/L inositol, 0.5mg/L nicotinic acid, 0.5mg/L pyridoxine hydrochloride, 0.1mg/L thiamine hydrochloride, 2mg/L glycine, 0.83mg/LKI, 6.2mg/LH₃BO₃, 22.3mg/LMnSO₄��4H₂O, 8.6mg/LZnSO₄��7H₂O, 0.25mg/L, Na₂MoO₄��2H₂O, 0.025mg/LCuSO₄��5H₂O, 0.025mg/LCoCl₂��6H₂O, 30g/L sucrose, 60g/LD-sorbitol, 1��3mg/Lpicloram, 0.5��1mg/L6-BA, with distilled water constant volume to 1L, pH value is 5.8;

(3) embedding: the yew cell in step (2) is embedded liquid with sodium alginate and mixes, instill Ca²⁺Solution is formed the glueballs comprising yew cell;

(4) pretreatment: the immobilization glueballs comprising yew cell that step (3) is obtained pretreatment in prepackage carrier fluid;

(5) load: take the pretreated glueballs of step (4) and use loading liquid to carry out vitrification process;

(6) freezen protective: take the glueballs after vitrification in step (5) processes, be immediately placed in the lump together with cryopreservation tube in liquid nitrogen after changing the same glass liquid of 4 DEG C of storages and preserve.

2. method according to claim 1, it is characterized in that, step (1) picking is successive transfer culture taxus callus inoculation after 15 days on solid medium, carrying out suspension culture under 22-25 DEG C of condition in 100-120 rev/min of constant-temperature table, callus inoculum concentration is the 5-10% of culture medium weight; Every 7-10 days successive transfer culture 1 time; Initial culture is used for the inoculation of step (2) after 15-20 days.

3. method according to claim 1 and 2, it is characterized in that, step (2) takes the finely disseminated Ramulus et folium taxi cuspidatae suspension cell obtained in step (1) and is inoculated in the MS-II fluid medium of improvement suspension culture 1 day, condition of culture is 22-25 DEG C, 100-120 rev/min of constant-temperature table is cultivated, and inoculum concentration is the 5-10% that the cell after sucking filtration step (1) is cultivated accounts for culture medium weight.

4. method according to claim 1, it is characterised in that step (3) sucking filtration also the yew cell after quality step (2) is cultivated such as weighs and mixes with sodium alginate embedding liquid, instills 0.1 mol/L Ca with aseptic rifle head²⁺Solution is formed the glueballs comprising yew cell.

5. the method according to claim 1 or 4, it is characterised in that in described step (3), sodium alginate embedding formula of liquid is: 3g/L sodium alginate, 180g/L glycerol, 1000mg/LKNO₃, 500mg/LKCl, 800mg/LNH₄NO₃, 170mg/LKH₂PO₄, 370mg/LMgSO₄��7H₂O, 37.3mg/LNa₂-EDTA, 27.8mg/LFeSO₄��7H₂O, 100mg/L inositol, 0.5mg/L nicotinic acid, 0.5mg/L pyridoxine hydrochloride, 0.1mg/L thiamine hydrochloride, 2mg/L glycine, 0.83mg/LKI, 6.2mg/LH₃BO₃, 22.3mg/LMnSO₄��4H₂O, 8.6mg/LZnSO₄��7H₂O, 0.25mg/LNa₂MoO₄��2H₂O, 0.025mg/LCuSO₄��5H₂O, 0.025mg/LCoCl₂��6H₂O, 30g/L sucrose, 1��3mg/Lpicloram, 0.5��1mg/L6-BA, with distilled water constant volume to 1L, pH value is 5.8.

6. method according to claim 1, it is characterised in that the prepackage carrier fluid of step (4) is LB7, formula is: 300g/L glycerol, 150g/L dimethyl sulfoxide, 400g/L sucrose, 150g/L trehalose, 2500mg/LKNO₃, 2500mg/LNH₄NO₃, 170mg/LKH₂PO₄, 370mg/LMgSO₄��7H₂O, 440mg/LCaCl₂��2H₂O, 37.3mg/LNa₂-EDTA, 27.8mg/LFeSO₄��7H₂O, 100mg/L inositol, 0.5mg/L nicotinic acid, 0.5mg/L pyridoxine hydrochloride, 0.1mg/L thiamine hydrochloride, 2mg/L glycine, 0.83mg/LKI, 6.2mg/LH₃BO₃, 22.3mg/LMnSO₄��4H₂O, 8.6mg/LZnSO₄��7H₂O, 0.25mg/LNa₂MoO₄��2H₂O, 0.025mg/LCuSO₄��5H₂O, 0.025mg/LCoCl₂��6H₂O, 1��3mg/Lpicloram, 0.5��1mg/L6-BA, with distilled water constant volume to 1L; The preloaded time is 30��60 minutes.

7. method according to claim 1, it is characterised in that step (5) uses loading liquid PVS3 to carry out vitrification and processes 30��60 minutes;PVS3 is with liquid MS minimal medium for solute, wherein containing the sucrose of the glycerol of mass percent 40% and mass percent 40%.

8. method according to claim 1, it is characterised in that thaw and the process of renewal cultivation is as follows:

Thaw: take the glueballs in step (6) and put into rapidly in 45 DEG C of waters and ceaselessly shake cryopreservation tube, within 1 minute, thawed;

Renewal cultivation: removing the vetrifying solution of the glue bead after thawing, soak glue bead 10 minutes by the MS culture medium of liquid, repeat 2 times, be inoculated in light culture on MS solid medium, once, cultivation temperature is 22 �� 1 DEG C to every 15 days successive transfer culture; Continue light culture at least 6 week.

9. method according to claim 1, it is characterized in that, taxus callus in described step (1) is that Taxus mairei is white or flaxen callus, or Ramulus et folium taxi cuspidatae is white or flaxen callus, or T. yunnanensis is white or flaxen callus, or Taxus media is white or flaxen callus.