CN104255705B - A method for overcoming the vitrification of regenerated seedlings after cryopreservation of Jerusalem artichoke stem tips - Google Patents

Abstract

The invention provides a method for overcoming the vitrification of regenerated seedlings after ultra-low temperature storage of Jerusalem artichoke stem tips. In the reagents used in the pre-cultivation stage, loading stage, PVS2 vitrification protection stage, and washing and unloading stage, fructose is used instead of sucrose to carry out ultra-low temperature Save processing. The method of the invention can effectively prevent the occurrence of vitrification after regeneration of the Jerusalem artichoke stem tip after ultra-low temperature preservation, the direct regeneration rate of the plant without callus can reach more than 40%, is easy to operate, and is suitable for ultra-low temperature preservation of the Jerusalem artichoke stem tip.

Description

A method for overcoming the vitrification of regenerated seedlings after cryopreservation of Jerusalem artichoke stem tips

technical field

The invention relates to a droplet vitrification ultra-low temperature preservation technology of plant germplasm resources, in particular to a method for overcoming the vitrification of regenerated seedlings after ultra-low temperature preservation of Jerusalem artichoke stem tips.

Background technique

Jerusalem artichoke (Helianthus tuberous L.) is a perennial herbaceous plant of the genus Sunflower in the Asteraceae family. It is an ecological and economical plant with high economic value, medicinal value, ornamental value and ecological value. It gets its name because it looks like a chrysanthemum on the ground and a taro on the ground. It is native to North America and widely distributed in the United States, Canada, Mexico and other countries. It was introduced into my country through Europe in the 17th century, and is distributed in the three northeastern provinces of my country, Inner Mongolia, Hebei, Henan and other provinces. Jerusalem artichoke is a vegetative crop with strong reproductive ability. Underground tubers reproduce at a rate of more than 20 times per year. Jerusalem artichoke has formed a large-scale production in Northwest my country. Jerusalem artichoke has wide ecological adaptability and strong stress resistance. Cold-resistant, drought-resistant, salt-alkali-resistant, windproof and sand control. Jerusalem artichoke root is rich in inulin, which is widely used in food and can be used as a sweetener and a raw material for the production of fructose. Jerusalem artichoke is called "the 21st century shared crop between humans and animals" by the Food and Agriculture Organization of the United Nations. The conversion of Jerusalem artichoke tubers into bioenergy such as ethanol and biofuel can be applied in chemical industry, biopharmaceutical, aerospace and other fields.

The ex situ conservation of plant germplasm resources can be divided into field preservation, test-tube seedling preservation and ultra-low temperature preservation. The germplasm resources of Jerusalem artichoke are currently preserved in the field and in test-tube seedlings in ex situ conservation. Because the underground tubers of Jerusalem artichoke are easily attacked by viruses, a large number of viruses accumulate, resulting in species degradation and reduced yield. Urban land development and environmental deterioration also seriously threaten the safe preservation of Jerusalem artichoke germplasm resources. Although the preservation of test-tube seedlings has the advantages of small area, less susceptible to natural disasters and pathogenic bacteria, less human and financial investment, and convenient germplasm resource exchange and transportation, it needs frequent subculture and renewal, and after multiple subcultures, it faces the Threats such as degradation or mutation. It has been proven that after a series of ultra-low temperature storage steps, plant tissue materials are stored in liquid nitrogen (under the condition of gas-phase liquid nitrogen or liquid-phase liquid nitrogen), and after thawing, new plants can still be regenerated under certain culture conditions, and maintain its genetic stability. It avoids the risk of germplasm loss or destruction caused by natural disasters in field preservation, and the risk of genetic trait variation or pollution caused by multiple subcultures in test-tube seedling tissue culture preservation.

The success criterion of the shoot tip cryopreservation method is that, after a series of cryopreservation treatments, the thawed shoot tip needs to be directly and successfully regenerated into normal seedlings after being regenerated and restored to culture under suitable conditions. However, in the stage of regeneration and recovery after cryopreservation, vitrification is prone to occur. Even if the stem tip of vitrification is enlarged, it is difficult to form regenerated seedlings, and regenerated seedlings are also prone to death. The vitrification phenomenon means that the regenerated seedlings become translucent, the leaves are light green or yellow-green, the leaves are thick, deformed, curled or shrunk easily, and the leaves are fragile and fragile. Anatomically, the leaves have no functional stomata, and due to the dysfunction of the guard cells, the stomata cannot be closed. The vitrified leaves do not have palisade tissue, only spongy tissue. Because the respiratory and photosynthetic functions of vitrified plants are not perfect, the differentiation ability of vitrified seedlings is low, it is difficult to subculture and reproduce, and it is even more difficult to transplant and survive.

The generation of vitrified seedlings after cryopreservation is related to many factors. Compared with the vitrification phenomenon of test-tube seedlings in tissue culture, the influencing factors are more complicated, and may involve various key steps in the cryopreservation process, and may also involve the regeneration recovery culture process Medium and culture environment conditions, such as water, light, nutritional conditions, hormone levels, etc. The cryopreservation process involves many key steps, such as pre-cultivation, loading, PVS2 vitrification protection, thawing, washing, etc. During the cryopreservation process, shoot tips and other materials may be subjected to different degrees of stress such as osmosis, dehydration, and chemical poisoning, resulting in some Cells lose their vitality or are damaged to varying degrees. If after thawing, washing and regeneration culture, the damage of some key cells has been repaired, it is beneficial to shoot tip regeneration and seedling formation. On the contrary, phenomena such as vitrification or callus may occur, which is not conducive to the successful regeneration of shoot tips.

Serious vitrification occurred in Jerusalem artichoke stem tips after cryopreservation by conventional methods. The researches on overcoming the vitrification phenomenon reported in the literature in the past mostly focused on overcoming or eliminating the vitrification phenomenon of the test-tube seedlings in the tissue culture process, and there were few studies on overcoming the vitrification phenomenon after cryopreservation. Overcoming the vitrification phenomenon after cryopreservation is one of the key links to solve the success of cryopreservation.

Contents of the invention

The purpose of the present invention is to provide a method for overcoming the vitrification of regenerated seedlings after cryopreservation of Jerusalem artichoke stem tips.

In order to realize the object of the present invention, a kind of method of the present invention overcomes the vitrification of regenerated seedlings after ultra-low temperature preservation of Jerusalem artichoke stem tip, and described method comprises the following steps:

1) Pre-cultivation: cutting the shoot tips of Jerusalem artichoke tissue cultured seedlings, and culturing in the dark at 25°C for 2-3 days in MS liquid medium containing 0.3-0.4mol/L fructose;

2) Loading: pre-cultured shoot tips were loaded in MS liquid medium containing 2.0mol/L glycerol and 0.4mol/L fructose at 25°C for 30-40min;

3) Treatment with PVS2 vitrification agent: transfer the loaded stem tip into the improved PVS2 vitrification agent for dehydration at 0°C for 15-40min;

4) Droplet vitrification method for cryopreservation: step 3) place the treated stem tip on an aluminum foil strip, and add the improved PVS2 vitrification protective agent to the stem tip, so that the stem tip is completely wrapped in the liquid formed by the protective agent. drop, put the aluminum foil strips in the cryotube, and then put them into liquid nitrogen for storage;

5) Thawing and washing: take out the cryotube from the liquid nitrogen, immerse the stem tip on the aluminum foil strip in MS culture solution containing 1.2mol/L fructose to thaw and wash at room temperature;

6) Step 5) After the treatment, the shoot tip is transferred to the recovery medium to recover the culture.

Wherein, the improved PVS2 vitrification agent described in steps 3) and 4) is MS liquid medium containing 30% glycerol, 15% dimethyl sulfoxide, 15% ethylene glycol and 0.4mol/L fructose.

The foregoing method, step 1) is specifically: take 4-5 weeks old Jerusalem artichoke tissue culture seedlings, peel off the outer leaves layer by layer, until the remaining 1-2 leaf primordia surround the shoot apex meristem, and cut off the shoot tip of 1.5-2mm , cultured in MS liquid medium containing 0.4mol/L fructose, 60rpm, 25°C in dark for 2-3 days.

In the aforementioned method, step 5) washes twice in total, 10 min each time, and washes with MS culture solution containing 1.2 mol/L fructose.

In the aforementioned method, the recovery medium described in step 6) is MS+0.1mg/LGA ₃ +15g/L sucrose, and the culture conditions are: after 3-5 days of dark culture in an incubator at 25°C, transfer to normal light conditions To cultivate.

The method provided by the invention is used to preserve and re-cultivate Jerusalem artichoke shoot tips, effectively avoiding the occurrence of vitrification, and the direct regeneration rate of plants without callus can reach more than 40%. The method provided by the invention is low in cost and easy to operate, and is an effective way to overcome the vitrification of regenerated seedlings after ultra-low temperature preservation of Jerusalem artichoke stem tips.

Description of drawings

Fig. 1 is the photograph of test-tube plantlet of Jerusalem artichoke in the embodiment 1 of the present invention.

Fig. 2 is a photo of Jerusalem artichoke shoot tip in Example 1 of the present invention.

Fig. 3 is a photo of vitrification after cryopreservation in Example 1 of the present invention.

Fig. 4 is a photo of seedlings regenerated directly without callus after cryopreservation in Example 2 of the present invention.

Detailed ways

The following examples are used to illustrate the present invention, but are not intended to limit the scope of the present invention. Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art, and the raw materials used are all commercially available products.

MS liquid medium: 30 grams of sucrose, 4.43 grams of MS medium powder (purchased from Beijing Ximeijie Technology Co., Ltd., model M519), pH 5.8, and dilute to 1 L.

MS liquid medium containing 0.4 mol/L fructose: 13.25 grams of fructose, 4.43 grams of MS medium powder (purchased from Beijing Ximeijie Technology Co., Ltd., model M519), pH 5.8, and the volume was adjusted to 1 L.

MS solid medium: 4.43 grams of MS medium powder (purchased from Beijing Ximeijie Technology Co., Ltd., model M519), add 30 grams of sucrose, and add 8.00 g of agar (purchased from Nangang District, Harbin, Heilongjiang). pH5.8, dilute to 1L.

Jerusalem artichoke tissue-cultured seedlings: the tubers of Jerusalem artichoke were collected in the reclamation section 20 of Xiaoshan District, Hangzhou City, Zhejiang Province after being inspected and collected in coastal areas by the Institute of Crop Science, Chinese Academy of Agricultural Sciences. The seedlings were stored in the National Germplasm Bank's Test Tube Seedling Bank, and the germplasm number is 2009331028.

Example 1 The phenomenon of vitrification of Jerusalem artichoke stem tips during ultra-low temperature storage

1. Reagent formulations

Pre-culture medium: MS liquid medium containing 0.4mol/L sucrose.

Loading solution: MS liquid medium containing 2.0mol/L glycerol and 0.4mol/L sucrose.

The preparation method of PVS2 vitrification protective agent: thoroughly mix 3.26mol glycerin, 2.42mol ethylene glycol, 1.9mol dimethyl sulfoxide and 0.4mol sucrose with MS liquid medium to 1L.

Unloading solution: MS liquid medium containing 1.20mol/L sucrose.

2. Ultra-low temperature preservation of Jerusalem artichoke stem tip

Get 4-5 week old Jerusalem artichoke tissue culture seedlings (Fig. 1), peel off the outer leaves layer by layer, until the remaining 1-2 leaf primordia surround the shoot apex meristem, cut off the 1.5-2mm shoot tip (Fig. 2), first Soak in the pre-culture medium for 3 days in the dark at 25°C (60rpm), then soak in the loading solution for 30min at 25°C (normal light), and finally dehydrate in PVS2 vitrification agent at 0°C for 15min (normal light). Put the stem tip after PVS2 dehydration on the aluminum foil strip (5mm × 20mm), add PVS2 solution dropwise to the stem tip, so that the stem tip is completely wrapped in the PVS2 solution droplet (add 4 drops on each aluminum foil strip) PVS2 vitrification agent, 16 microliters per drop, accommodate 5 shoot tips), and finally transfer the aluminum foil strips to a cryopreservation tube filled with liquid nitrogen, and put them into liquid nitrogen for storage. Take out the cryopreservation tube from the liquid nitrogen, put the aluminum foil strip into the MS unloading solution of 1.20mol/L sucrose and thaw at room temperature for 20min, change the washing solution every 10min, and then transfer the shoot tip to the recovery medium (MS +0.1mg/L GA ₃ +15g/L sucrose), placed in a 25°C incubator for dark cultivation for 3-5 days, and then transferred to normal light conditions for cultivation.

3. Survival rate and regeneration rate statistics

The survival rate (the number of surviving plants÷the number of shoot tips×100%) was counted after one week of recovery culture, and the regeneration rate was counted two weeks later (the number of regenerated plants÷the number of shoot tips×100%).

The survival rates of the three repeated experiments were 92.31%, 92.86%, 92.86%, and the average survival rate was 92.67%. The regeneration rates of the three repeated experiments were 69.23%, 92.86%, and 85.71%, respectively, and the average regeneration rate was 82.60%. Although the survival rate and regeneration rate were high, severe vitrification occurred in the regenerated seedlings after cryopreservation (Fig. 3).

Embodiment 2 The overcoming of the vitrification phenomenon of Jerusalem artichoke stem tip cryopreservation

1. Reagent formulations

Pre-culture solution containing fructose: MS liquid medium containing 0.4mol/L fructose (substitute sucrose with fructose).

Mannitol-containing pre-culture solution: MS liquid medium containing 0.4mol/L mannitol (replacing sucrose with mannitol).

Loading solution containing fructose: MS liquid medium containing 2.0 mol/L glycerol and 0.4 mol/L fructose (substitute sucrose with fructose).

Loading solution containing mannitol: MS liquid medium containing 2.0mol/L glycerol and 0.4mol/L mannitol (mannitol is used instead of sucrose).

Preparation method of fructose-containing vitrification agent: 3.26mol glycerin, 2.42mol ethylene glycol, 1.9mol dimethyl sulfoxide and 0.4mol fructose were thoroughly mixed with MS liquid medium to 1L (sucrose was replaced by fructose).

The preparation method of the vitrification protective agent containing mannitol: fully mix 3.26mol glycerin, 2.42mol ethylene glycol, 1.9mol dimethyl sulfoxide and 0.4mol mannitol with MS liquid medium to 1L (replace with mannitol sucrose).

Fructose-containing unloading solution: MS liquid medium containing 1.2mol/L fructose (substitute sucrose with fructose).

Unloading solution containing mannitol: MS liquid medium containing 1.2mol/L mannitol (mannitol is used instead of sucrose).

2. Ultra-low temperature preservation of Jerusalem artichoke stem tip

Scheme 1: The cryopreservation process is the same as in Example 1, and all technical steps use fructose-containing reagents instead of sucrose reagents.

Scheme 2: The cryopreservation process is the same as in Example 1, the pre-culture solution is replaced with a reagent containing fructose, and the reagents used in other technical steps are still reagents containing sucrose.

Scheme 3: The cryopreservation process is the same as in Example 1, and all technical steps use mannitol-containing reagents instead of sucrose reagents.

Scheme 4: The cryopreservation process is the same as in Example 1, the pre-culture solution is replaced with a reagent containing mannitol, and the reagents used in other technical steps are still reagents containing sucrose.

3. Survival rate and regeneration rate statistics

The statistical methods of survival rate and regeneration rate are the same as in Example 1, and the results are shown in Table 1.

Table 1 Statistical results of survival rate and regeneration rate

As can be seen from the above statistical results, after all the steps in the cryopreservation technical process of Jerusalem artichoke stem tips in Scheme 3 are replaced by mannitol-containing reagents containing sucrose, although the regeneration rate can be maintained at 45%, there are still 2 in the regenerated seedlings. /3 Vitrification occurs. After replacing the sucrose-containing reagent with the fructose-containing reagent in all steps of Scheme 1, although the regeneration rate was reduced (40%), all the regenerated seedlings were healthy regenerated seedlings, and there was no vitrification phenomenon, and the vitrification phenomenon of the regenerated seedlings was overcome (Figure 4).

Although the present invention has been described in detail with general descriptions and specific embodiments above, it is obvious to those skilled in the art that some modifications or improvements can be made on the basis of the present invention. Therefore, the modifications or improvements made on the basis of not departing from the spirit of the present invention all belong to the protection scope of the present invention.

Claims (3)

1. a method for overcoming the vitrification of regenerated seedlings after ultra-low temperature preservation of Jerusalem artichoke stem tip, is characterized in that, comprises the following steps: 1) Pre-cultivation: cutting the shoot tips of Jerusalem artichoke tissue cultured seedlings, and culturing in the dark at 25°C for 2-3 days in MS liquid medium containing 0.3-0.4mol/L fructose; 2) Loading: pre-cultured shoot tips were loaded in MS liquid medium containing 2.0mol/L glycerol and 0.4mol/L fructose at 25°C for 30-40min; 3) Treatment with PVS2 vitrification agent: transfer the loaded stem tip into the improved PVS2 vitrification agent for dehydration at 0°C for 15-40min; 4) Droplet vitrification method for cryopreservation: step 3) place the treated stem tip on an aluminum foil strip, and add the improved PVS2 vitrification protective agent to the stem tip, so that the stem tip is completely wrapped in the liquid formed by the protective agent. drop, put the aluminum foil strips in the cryotube, and then put them into liquid nitrogen for storage; 5) Thawing and washing: take out the cryotube from the liquid nitrogen, immerse the stem tip on the aluminum foil strip in MS culture solution containing 1.2mol/L fructose to thaw and wash at room temperature; 6) Step 5) After the treatment, the shoot tip is transferred to the recovery medium for recovery; Wherein, the improved PVS2 vitrification agent described in steps 3) and 4) is MS liquid medium containing 30% glycerol, 15% dimethyl sulfoxide, 15% ethylene glycol and 0.4mol/L fructose; The recovery medium described in step 6) is MS + 0.1 mg/L GA ₃ + 15 g/L sucrose, and the culture conditions are as follows: after 3-5 days of dark culture in an incubator at 25°C, the culture is transferred to normal light conditions. 2. The method according to claim 1, characterized in that, step 1) is specially: get the Jerusalem artichoke tissue culture seedling of 4-5 week seedling age, peel off the outer layer of leaves layer by layer, until the remaining 1-2 leaf primordia surround the stem tip For the meristem, the shoot tip of 1.5-2 mm was cut out, and cultured in MS liquid medium containing 0.4 mol/L fructose at 60 rpm at 25° C. for 2-3 days in the dark. 3. The method according to claim 1, characterized in that, step 5) washes 2 times in total, each 10 min, and washes with MS culture fluid containing 1.2mol/L fructose.