CN113331052A - Process for cultivating superior blueberry products by using micro-freezing biotechnology - Google Patents

Abstract

The invention relates to a process for cultivating superior blueberry products by using a micro-freezing biotechnology. In order to solve the problems of difficult rooting, low survival rate and the like of test-tube plantlets in the conventional blueberry tissue culture, the method creatively combines a micro-freezing technology with the blueberry tissue culture, and performs micro-freezing treatment on blueberry explants before blueberry tissue culture.

Description

Process for cultivating superior blueberry products by using micro-freezing biotechnology

Technical Field

The invention relates to the field of blueberry cultivation, in particular to a process for cultivating superior blueberry products by using a micro-freezing biotechnology.

Background

Blueberries (blueberries) are perennial deciduous leaves or evergreen shrubs or shrubs of Vaccinium of Ericaceae (Ericaceae), and the fruits are blue berries and rich in various physiologically active ingredients such as anthocyanin and flavone, so that the Blueberry extract has high medicinal value and health care effect. Compared with grafting, the tissue culture method has the advantages of capability of obtaining virus-free seedlings, high propagation speed, no limitation of seasons, capability of obtaining a large number of propagules in a short period, capability of growing seedlings younger than conventional seedlings in stage development and the like, so that the tissue culture technology is more and more widely applied to blueberry seedling breeding. However, the problems of difficult rooting, low rooting rate and the like of test-tube plantlets are easy to occur during blueberry tissue culture.

The micro-freezing technology is to directly freeze products such as fruits, vegetables and meat under the mediation of a micro-freezing solution, but cell membranes of the products are not frozen and are in a micro-freezing state of organisms, so that the fresh and alive quality of the frozen products is maintained. The inventor creatively applies the micro-freezing technology to crop breeding, solves the problems existing in the crop breeding through the micro-freezing biotechnology, and finds that the rice seeds subjected to micro-freezing treatment have better budding and growth in the micro-freezing method and the application of the patent CN 202110073639.2.

In order to solve the problems of difficult rooting, low rooting rate, low survival rate and the like of test-tube plantlets in the conventional blueberry tissue culture, the micro-freezing technology is creatively combined with the blueberry tissue culture, and the blueberry explant is subjected to micro-freezing treatment before the blueberry tissue culture, so that the rooting capacity and the proliferation capacity of the explant during subsequent tissue culture are enhanced, and the antiviral property of the explant is also improved.

Disclosure of Invention

The invention discloses a method for processing a blueberry explant, which comprises the following specific steps:

1) selecting a blueberry explant: taking robust blueberry twigs or semi-lignified young shoots;

2) micro-freezing treatment: carrying out micro-freezing treatment at-6 to-2 ℃ for 30 to 40 minutes;

3) and (3) gradually thawing: the temperature is raised by 1-5 ℃ every 15-30 minutes.

Further, the micro-freezing treatment is carried out at-4 ℃ for 30-40 minutes.

Further, gradual thawing: the temperature is raised to 2 ℃ every 20 minutes until the temperature is normal.

The normal temperature in the application can be outdoor temperature or 10-30 ℃, and preferably, the normal temperature is 15-25 ℃.

The blueberry explant can also be leaf with or without petioles and stem segments of current-year green branches.

The blueberry explant in the application can be a robust blueberry twig, a semi-lignified young shoot, a blueberry stem segment or stem tip, an annual green branch stem segment, a leaf with a petiole, a leaf without a petiole and the like.

A blueberry explant is obtained by processing through the method.

A culture method of blueberry tissue culture seedlings comprises a treatment method of the blueberry explants.

Further, the culture method further comprises:

explant disinfection: cutting the explant subjected to the micro-freezing treatment into stem sections, cleaning and disinfecting;

and (3) induction culture: inoculating the blueberry seeds into an induction culture medium for induction differentiation to obtain blueberry primary tissue culture seedlings;

rooting culture: inoculating to a rooting culture medium for rooting culture to obtain a rooting test-tube plantlet.

The explant sterilization in the application comprises explant sterilization, and can be performed by adopting modes of ethanol, mercuric chloride, sodium hypochlorite, hydrogen peroxide and the like, and the explant can be washed for a plurality of minutes by running water after being soaked in laundry powder liquid and the like before sterilization. The explant is disinfected by ethanol, the use concentration of the explant is generally 70-75%, and the time is 30s-10 min; the mercury bichloride is used at 0.1% concentration for 5-10 min. Explant sterilization may also be a combination of two or more of the above-mentioned several means, such as a combination of ethanol and mercuric chloride, as specified: sterilizing with 70% ethanol for 1-2min, sterilizing with 0.1% mercuric chloride for 6-10min, and washing with sterile water for several times. The number of times may vary from 1 to 10 times.

The culture medium can be selected from various culture media, such as WPM, MS, 1/2WPM, 1/2MS or MW culture medium between WPM and MS culture medium;

preferably, the induction medium is an MS medium;

preferably, hormones such as ZT, IBA, 6-BA, NAA, etc. can be added into the induction medium; more preferably, the concentration of the hormone can be selected within the range of 0.5-2.0 mg/L of ZT, 0.5-5.0 mg/L of 6-BA and 0.05-0.3 mg/L of NAA;

preferably, the suitable temperature is 23-25 ℃ and the illumination intensity is 1600LX-2500 LX.

Further, carrying out subculture after induction culture, and then carrying out rooting culture, wherein the subculture is to take stems or leaves of blueberry primary tissue culture seedlings obtained by induction culture, carry out subculture in a subculture medium, and obtain blueberry tissue culture seedlings after 3-7 times of subculture;

preferably, the stem tip of the blueberry primary tissue culture seedling is cut into about 1-3cm or the leaves of the tissue culture seedling are placed in a subculture medium for subculture;

preferably, cytokinins and/or auxins are added to the secondary culture medium; preferably, ZT, IBA, GA or the combination of the hormones is added into the subculture medium; the concentration of the hormone can be selected within the range of 0.5-4.0 mg/L of ZT and 0.05-1.0 mg/L of 6-BA;

more preferably, the subculture medium is WPM + ZT 0.5-1.0 mg/L.

Preferably, hormones, such as IBA, NAA or a combination of the hormones, are added to the rooting medium; the preferable hormone concentration selectable range is IBA 0.05-3.0 mg/L, NAA 0.05-2.0 mg/L;

more preferably, the rooting medium is 1/2WPM, 6% agar, 2% sucrose, 0.2% activated carbon and 0.5-2.0 mg/L IBA.

Further, after the rooting test-tube seedlings are obtained, ex-vitro rooting is carried out, wherein ex-vitro rooting is realized by cutting the rooting seedlings into small segments and cutting the small segments into a rooting matrix to obtain blueberry seedlings.

Preferably, after the rooting test-tube seedling is placed in a transplanting environment for several days, a bottle opening is opened, seedling training is carried out, the rooting test-tube seedling can be transplanted to a greenhouse for plug cultivation after 3-5 days of seedling training, and a plant can continuously grow new roots after being transplanted outside the bottle.

Further, the explant sterilization is that the explant after the micro-freezing treatment is cut into 0.5-3cm stem sections and is cleaned, the explant is sterilized by adopting methods including 70-75% alcohol, mercuric chloride, sodium hypochlorite, hydrogen peroxide and the like, and the sterilized explant is washed by sterile water for a plurality of times;

the induction culture is to inoculate the sterilized explants on an MS induction culture medium for induction differentiation to obtain blueberry primary tissue culture seedlings;

the rooting culture is to take more than 1cm of stem tips to be inoculated into an improved 1/2WPM + 6% agar + 2% sucrose + 0.2% active carbon + IBA 0.5-2.0 mg/L rooting culture medium for rooting culture.

A culture method of blueberry tissue culture seedlings comprises the following steps:

selecting a blueberry explant: taking robust blueberry twigs, semi-lignified new shoots, leaves or current-year green branch stems;

micro-freezing treatment: carrying out micro-freezing treatment at-6 to-2 ℃ for 30 to 40 minutes;

explant disinfection: cutting the explant subjected to the micro-freezing treatment into stem segments, cleaning and then sterilizing, wherein the sterilization comprises the steps of placing the stem segments on an aseptic workbench, soaking the stem segments in 70-75% alcohol for 1-2min, sterilizing for 6-10min by 0.1% mercuric chloride, and washing with aseptic water for several times;

and (3) induction culture: inoculating the sterilized explants into an MS induction culture medium for induction differentiation to obtain blueberry primary tissue culture seedlings;

subculturing: taking stems or leaves of blueberry primary tissue culture seedlings obtained by induction culture, cutting stem sections into about 1-3cm, carrying out subculture in a WPM + ZT 0.5-1.0 mg/L subculture medium, and carrying out subculture for 3-7 times to obtain blueberry tissue culture seedlings;

rooting culture: and (3) inoculating the blueberry tissue culture seedling into 1/2WPM, 6% agar, 2% cane sugar, 0.2% activated carbon and IBA 0.5-2.0 mg/L rooting culture medium for rooting culture to obtain a rooting test-tube seedling.

The invention has the advantages that:

the blueberry explant after the micro-freezing treatment has stronger rooting capacity, and the performance of the stages of rooting culture, ex-vitro rooting and the like is far better than that of the blueberry explant which is not subjected to the micro-freezing treatment.

The tissue culture seedling obtained from the blueberry explant after the micro-freezing treatment shows certain antiviral capacity, because the cells start an adverse resistance mechanism under the influence of the micro-freezing environment during the micro-freezing treatment.

Drawings

FIG. 1 shows blueberry seedlings in subculture;

FIG. 2 is a comparison graph of blueberry tissue culture with and without partial freezing.

Detailed Description

The present invention is further illustrated below with reference to specific examples, which are intended to be illustrative only and are not to be construed as limiting the invention.

Example blueberry explant partial freezing treatment and tissue culture

Materials: the blueberry variety is Bridgy Tower, and the micro-freezer and the micro-freezing liquid are purchased from flourishing blueberry group, Inc.;

preparation of a culture medium: the induction culture medium is MS culture medium, the subculture medium is WPM + ZT 0.5-1.0 mg/L, the rooting culture medium is 1/2WPM + 6% agar + 2% sucrose + 0.2% active carbon + IBA 0.5-2.0 mg/L, and the mixture is sterilized for later use.

And (3) blueberry explant treatment and tissue culture:

(1) selecting a blueberry explant: taking robust blueberry twigs or semi-lignified young shoots;

(2) micro-freezing treatment: slightly freezing at-4 deg.C for 30-40 min (the control group did not);

(3) explant disinfection: cutting the material into 1-2cm stem segments, sterilizing with 70% alcohol for 1-2min, sterilizing with 0.1% mercuric chloride for 6-10min, and washing with sterile water for 3-7 times;

(4) and (3) induction culture: inoculating the disinfected stem section of the Bridgkin's pagoda into an MS induction culture medium for culture, wherein the temperature is 23-25 ℃, the illumination intensity is 1600LX-2500LX, the sprouting condition of the explant is observed 1 week after inoculation, and the sprouting rate of the explant is counted after 40 days;

(5) subculturing: cutting the cluster buds cultured by induction into 1-2cm stem sections, inoculating 30 in each group, transferring to a subculture medium (WPM + ZT 0.5-1.0 mg/L) for subculture, and counting the multiplication coefficient of the blueberries, wherein the branch height is 2-7cm every 30-40d generation;

(6) rooting culture: inoculating more than 2.5cm of new branches or young shoots into an improved 1/2WPM rooting culture medium (1/2WPM + 6% agar + 2% sucrose + 0.2% activated carbon + IBA 0.5-2.0 mg/L) for rooting culture, culturing 3 plants in each bottle, after inoculation, firstly performing dark culture for 10 days, then transferring to the light for culture at the culture temperature of 20-25 ℃, starting rooting in about 2 weeks generally, and counting the rooting rate at 40 days.

(7) Rooting outside the bottle: putting bottle seedlings of rooted seedlings in a room with good light, uncovering, hardening the seedlings for 3-5 days, removing a culture medium, transferring the bottle seedlings to a transplanting medium of a greenhouse for plug culture, wherein the transplanting medium consists of turfy soil, perlite and moss, shading properly, paying attention to fertilizer and water management, investigating the transplanting survival rate of the transplanted blueberry plants after 40 days, and transferring the bottle seedlings to the outside of the greenhouse for strong seedling culture after 60 days.

Germination rate (%) — number of sprouts/number of inoculated sprouts

Proliferation factor is the number of proliferated stem/number of inoculated stem

Rooting ratio (%) (number of rooted seedlings/number of inoculated seedlings)

Survival rate (%) of transplantation/number of transplanted plants

The inventor repeatedly searches and optimizes the combination and concentration of the subculture medium, finally determines that the optimal WPM + ZT is 0.5-1.0 mg/L, and finishes the breeding of the subculture seedlings of a plurality of blueberry varieties, wherein the number of the subculture seedlings reaches 1.5 ten thousand, and the condition of partial subculture seedlings is shown in figure 1, so that the subculture seedlings grow well. The result after 40 days of transplantation is shown in fig. 2, the upper graph in fig. 2 is a slightly frozen treatment group, the lower graph is a group which is not slightly frozen, and specific statistical results are shown in the following table; the difference between the proliferation coefficients of the two groups is not large; the blueberry test tube has slow rooting and low rooting rate, and the inventor determines that the optimal rooting culture medium is 1/2WPM + 6% agar + 2% sucrose + 0.2% active carbon + IBA 0.5-2.0 mg/L through repeated tests, and even if the group is not subjected to micro-freezing treatment, 30 young shoots are inoculated, after 40 days of culture, the number of rooted plants is 20, the rooting rate of blueberries is 67%, the group is subjected to micro-freezing treatment, 30 young shoots are inoculated, after 40 days of culture, the number of rooted plants is 28, and the rooting rate of blueberries is as high as 93%. After 40 days of transplanting, the survival rate of the blueberry plants is counted, 30 plants in the slightly frozen group survive and grow well, and 13 plants in the group which does not undergo the slightly frozen treatment survive but grow poorly.

TABLE 1

Grouping	Micro-freezing treatment group	Non-micro-freezing treatment group
			Germination percentage (%)	93％	73％
Coefficient of proliferation	8.1	7.6
			Rooting percentage (%)	93％	67％
Survival Rate of transplantation (%)	100％	43.3％

By adopting the system and the process provided by the application to carry out blueberry tissue culture, different varieties of blueberries (such as Lanfeng, Duke, Meideng and the like) can adapt to the blueberry tissue culture, the blueberry tissue culture has the characteristics of high rooting speed, large hairy root number and high rooting rate, and the problems that the blueberry industrialization development is restricted by difficult hairy root, long required time and the like in the blueberry production process are solved.

After 60 days, the blueberry plants are moved to the outside of the greenhouse for strong seedling cultivation, and the blueberry plants subjected to micro-freezing treatment have stronger antiviral ability compared with the blueberry plants not subjected to micro-freezing treatment in the cultivation process outside the greenhouse, because the cells start an anti-adversity mechanism under the influence of the micro-freezing environment during the micro-freezing treatment, the blueberry plants subjected to tissue culture show certain antiviral ability.

Those of ordinary skill in the art will understand that: various changes, modifications, substitutions and alterations can be made to the embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the claims and their equivalents. The following examples are examples of experimental methods not indicating specific conditions, and the detection is usually carried out according to conventional conditions or according to the conditions recommended by the manufacturers.

Claims (10)

1. A method for processing a blueberry explant comprises the following specific steps:

1) selecting a blueberry explant: taking robust blueberry twigs or semi-lignified young shoots;

2) micro-freezing treatment: carrying out micro-freezing treatment at-6 to-2 ℃ for 30 to 40 minutes;

3) and (3) gradually thawing: the temperature is raised by 1-5 ℃ every 15-30 minutes.

2. The process according to claim 1, wherein the partial freezing is carried out at-4 ℃ for 30 to 40 minutes.

3. The process according to claim 1, characterized in that the gradual thawing: the temperature is raised to 2 ℃ every 20 minutes until the temperature is normal.

4. The treatment method as claimed in any one of claims 1-2, wherein the blueberry explant is leaf with or without petiole, and current-year green shoot stem segment.

5. A culture method of blueberry tissue culture seedlings, which is characterized by comprising a treatment method of blueberry explants according to any one of claims 1 to 3.

6. The culture method according to claim 5, further comprising:

explant disinfection: cutting the explant subjected to the micro-freezing treatment into stem sections, cleaning and disinfecting;

and (3) induction culture: inoculating the blueberry seeds into an induction culture medium for induction differentiation to obtain blueberry primary tissue culture seedlings;

rooting culture: inoculating to a rooting culture medium for rooting culture to obtain a rooting test-tube plantlet.

7. The treatment method according to claim 6, wherein the induction culture is followed by subculture and then rooting culture, wherein the subculture is to take stems or leaves of primary tissue culture seedlings of blueberries obtained by the induction culture and carry out subculture in a subculture medium, and the tissue culture seedlings of blueberries are obtained after 3-7 times of subculture; preferably, cutting the stem tip of the blueberry primary tissue culture seedling into about 1-3 cm; preferably, cytokinins and/or auxins are added to the secondary culture medium; more preferably, the subculture medium is WPM + ZT 0.5-1.0 mg/L.

8. The method according to any one of claims 5 to 7, wherein ex vitro rooting is performed after the rooting test-tube plantlet is obtained, wherein ex vivo rooting is performed by cutting the rooting plantlet into small segments and cutting the small segments into a rooting matrix to obtain blueberry plantlets.

9. The method as claimed in claim 6, wherein the explant sterilization is that the explant after the micro-freezing treatment is cut into 0.5-3cm stem sections and cleaned, sterilized by methods including 70-75% alcohol, mercuric chloride, sodium hypochlorite, hydrogen peroxide and the like, and washed by sterile water for several times after sterilization; the induction culture is to inoculate the sterilized explants on an MS induction culture medium for induction differentiation to obtain blueberry primary tissue culture seedlings; the rooting culture is to take more than 1cm of stem tips to be inoculated into an improved 1/2WPM + 6% agar + 2% sucrose + 0.2% active carbon + IBA 0.5-2.0 mg/L rooting culture medium for rooting culture.

10. The method of claim 5, comprising the steps of:

selecting a blueberry explant: taking robust blueberry twigs, semi-lignified new shoots, leaves or current-year green branch stems;

micro-freezing treatment: carrying out micro-freezing treatment at-6 to-2 ℃ for 30 to 40 minutes;

explant disinfection: cutting the explant subjected to the micro-freezing treatment into stem segments, cleaning and then sterilizing, wherein the sterilization comprises the steps of placing the stem segments on an aseptic workbench, soaking the stem segments in 70-75% alcohol for 1-2min, sterilizing for 6-10min by 0.1% mercuric chloride, and washing with aseptic water for several times;

and (3) induction culture: inoculating the sterilized explants into an MS induction culture medium for induction differentiation to obtain blueberry primary tissue culture seedlings;

subculturing: taking stems or leaves of blueberry primary tissue culture seedlings obtained by induction culture, cutting stem sections into about 1-3cm, carrying out subculture in a WPM + ZT 0.5-1.0 mg/L subculture medium, and carrying out subculture for 3-7 times to obtain blueberry tissue culture seedlings;

rooting culture: and (3) inoculating the blueberry tissue culture seedling into 1/2WPM, 6% agar, 2% cane sugar, 0.2% activated carbon and IBA 0.5-2.0 mg/L rooting culture medium for rooting culture to obtain a rooting test-tube seedling.

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