CN114287341A - Method for culturing blueberry tissue - Google Patents
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Abstract
The invention discloses a method for culturing blueberry tissues, wherein the blueberry is a jewel, and the method comprises the following steps: selecting explants: taking the young branch of the current-year blueberry, and shearing the stem with the bud; and (3) disinfection treatment of explants: disinfecting the stem section with the buds by using alcohol, disinfecting the stem section with the bud by using mercury bichloride, and washing the stem section for later use to obtain a disinfected stem section with the buds; primary culture: cutting the sterilized stem section with buds into single stem sections with buds, and inserting the lower ends of the single stem sections into a primary culture medium for primary culture; and (3) proliferation culture: inoculating the single stem after primary culture into a proliferation culture medium for proliferation culture; rooting culture: and inoculating the single stem section after propagation culture into a rooting culture medium for rooting culture. By definitely and reasonably setting the hormone content in the primary culture medium, the enrichment culture medium and the rooting culture medium and reasonably sterilizing the single stem section before inoculation, the tissue culture survival rate of the blueberries is high and the tissue culture is easy to succeed.
Description
Technical Field
The invention relates to tissue culture of plants, in particular to a method for culturing blueberry tissues.
Background
The blueberry is a plant of Vaccinium of Ericaceae, mature fruit is blue, seed is small, and the blueberry is fresh and pleasant and has fine pulp. Blueberry has extremely high nutritive value, is rich in various vitamins (VA, VB, VC and VE), various mineral elements (K, Fe, Zn and Ca), protein, anthocyanin, edible fiber and the like, and has unique effects of improving immunity, improving eyesight, resisting cancer, improving circulation, softening blood vessels, delaying nerve aging, relieving eye fatigue, preventing senescence, resisting mutation and the like, so the blueberry is known as 'fruit queen'. Because the fruits contain various polyphenol physiological active ingredients such as anthocyanin and flavone, the fruit has various physiological active functions of promoting resynthesis of visual pigment, resisting inflammation, improving immunity, resisting cardiovascular diseases, resisting aging, preventing cancer and the like, is praised as '21 st century functional health-care berry', is listed as one of five kinds of human health foods by international food and agriculture organizations, and the fruits and products thereof are in shortage in the world.
Since the 1999 commercial cultivation in China, over 10 provinces in China begin the commercial cultivation of blueberries. Domestic industrial and commercial enterprises and international financial capital participate in the blueberry industry, so that blueberries become the emerging fruit trees which are developed most rapidly in China at present. With the continuous increase of cultivation area and yield, related application research and application basic research are receiving more and more attention.
Tissue culture is carried out on horticultural crops, researches on blueberries are also carried out at present, but the regeneration rate of some varieties is low, and a more key problem exists is that the culture of tissue seedlings of blueberries is not easy to succeed and the survival rate is relatively low.
Disclosure of Invention
The invention aims to provide a method for culturing blueberry tissues, and solves the problems that the hormone proportion in a culture medium for blueberry tissue culture is not clear, and single stem sections for tissue culture are not reasonably disinfected, so that the survival rate of blueberry tissue culture seedlings is low, and the culture is not easy to succeed.
In order to achieve the purpose, the invention provides a method for culturing blueberry tissues, wherein the blueberry is a jewel, and the method is characterized by comprising the following steps:
(1) selecting explants: taking the young branch of the current-year blueberry, and shearing the stem with the bud;
(2) and (3) disinfection treatment of explants: disinfecting the stem section with the buds by using alcohol, then disinfecting by using mercury bichloride, and then washing for later use;
(3) primary culture: cutting the stem section with the buds into single stem sections with buds, and inserting the lower ends of the single stem sections into a primary culture medium for primary culture;
(4) and (3) proliferation culture: inoculating the single stem after primary culture into a proliferation culture medium for proliferation culture;
(5) rooting culture: inoculating the single stem after propagation culture into a rooting culture medium for rooting culture;
wherein the primary culture medium is a WPM culture medium, the proliferation culture medium is a WPM culture medium, and the rooting culture medium is 1/2WPM culture medium.
In the technical scheme, the blueberry tissue culture method is characterized in that the blueberry is a jewel, and the tissue culture survival rate of the blueberry is high and the blueberry is easy to succeed by clearly and reasonably setting the hormone content in the primary culture medium, the proliferation culture medium and the rooting culture medium and carrying out reasonable sterilization treatment on single stem sections before inoculation.
Additional features and advantages of the invention will be set forth in the detailed description which follows.
Drawings
The accompanying drawings, which are included to provide a further understanding of the invention and are incorporated in and constitute a part of this specification, illustrate embodiments of the invention and together with the description serve to explain the principles of the invention and not to limit the invention. In the drawings:
FIG. 1 a is a schematic diagram of the growth of a primary culture of a "jewellery" variety in MS medium; b is a growth schematic diagram of primary culture of the jewelry variety in a WPM culture medium;
FIG. 2 a is a schematic representation of the growth of a "jewellery" variety in subculture in MS medium; b is a growth schematic diagram of subculture of the jewelry variety in a WPM medium;
FIG. 3 a is a schematic representation of rooting in the subculture of the "jewellery" variety in 1/2MS medium; b is a rooting diagram of subculture of the jewelry variety in 1/2WPM medium.
Detailed Description
The following describes in detail specific embodiments of the present invention. It should be understood that the detailed description and specific examples, while indicating the present invention, are given by way of illustration and explanation only, not limitation.
The endpoints of the ranges and any values disclosed herein are not limited to the precise range or value, and such ranges or values should be understood to encompass values close to those ranges or values. For ranges of values, between the endpoints of each of the ranges and the individual points, and between the individual points may be combined with each other to give one or more new ranges of values, and these ranges of values should be considered as specifically disclosed herein.
The invention provides a tissue culture method of blueberry variety 'jewelry', which is characterized by comprising the following steps:
(1) selecting explants: taking the young branch of the current-year blueberry, and shearing the stem with the bud;
(2) and (3) disinfection treatment of explants: disinfecting the stem section with the buds by using alcohol, then disinfecting by using mercury bichloride, and then washing for later use;
(3) primary culture: cutting the stem section with the buds into single stem sections with buds, and inserting the lower ends of the single stem sections into a primary culture medium for primary culture;
(4) and (3) proliferation culture: inoculating the single stem after primary culture into a proliferation culture medium for proliferation culture;
(5) rooting culture: inoculating the single stem after propagation culture into a rooting culture medium for rooting culture.
In the present invention, the kind of the medium can be selected in a wide range, but in order to further improve the survival rate of tissue culture, it is preferable that the primary medium is WPM medium, the multiplication medium is WPM medium, and the rooting medium is 1/2WPM medium.
In step (1) of the present invention, the length of the budded stem segment may be selected within a wide range, but in order to further improve the survival rate of tissue culture, it is preferable that the length of the budded stem segment is 3 to 4 cm.
In step (2) of the present invention, the conditions for alcohol sterilization may be selected within a wide range, but in order to further improve the survival rate of tissue culture, it is preferable that the alcohol sterilization is: soaking the stem with bud in 70-75% alcohol, and sterilizing for 20-40s under shaking.
In step (2) of the present invention, the conditions for mercuric chloride disinfection can be selected within a wide range, but in order to further improve the survival rate of tissue culture, it is preferable that the mercuric chloride disinfection is: soaking the stem segments with buds sterilized by the alcohol into mercuric chloride with the mass fraction of 0.08-0.12%, and sterilizing for 8-10min by shaking.
In step (2) of the present invention, the washing conditions can be selected within a wide range, but in order to prevent the germinated stem segments from secondary contamination and to avoid mercury and alcohol residues in the germinated stem segments before inoculation, it is preferable that the washing is performed by aseptic washing for 5 to 6 times and for 1 to 3min each time.
In step (3) of the present invention, in order to prevent the introduction of new contaminating bacteria during inoculation, it is preferable that the inoculation of the primary medium is performed around an alcohol lamp.
In step (3) of the present invention, the length of the single-stem segment may be selected within a wide range, but in order to further improve the survival rate of the tissue culture, it is preferable that the length of the single-stem segment is 1-2 cm.
In step (3) of the present invention, in order to further improve the survival rate of tissue culture, preferably, the primary medium further contains zeatin and indolebutyric acid.
In step (3) of the present invention, the content of zeatin and indolebutyric acid can be selected within a wide range, but in order to further improve the survival rate of tissue culture, it is preferable that the content of zeatin is 3-5mg and the content of indolebutyric acid is 0.3-0.5mg with respect to 1L of the primary medium.
In step (3) of the present invention, in order to further avoid the existence of mercuric chloride and alcohol in the budded stem segment before inoculation, it is preferable that the single stem segment is subjected to surface moisture absorption by using sterile filter paper.
In step (4) of the present invention, in order to further improve the survival rate of tissue culture, preferably, the propagation medium further contains zeatin and indolebutyric acid.
In step (4) of the present invention, the content of zeatin and indolebutyric acid can be selected within a wide range, but in order to further improve the survival rate of tissue culture, it is preferable that the content of zeatin is 1-3mg and the content of indolebutyric acid is 0.1-0.3mg with respect to 1L of the propagation medium.
In step (5) of the present invention, in order to further improve the survival rate of tissue culture, it is preferable that the rooting medium further contains indolebutyric acid.
In step (5) of the present invention, the content of indolebutyric acid can be selected within a wide range, but in order to further improve the survival rate of tissue culture, it is preferable that the content of indolebutyric acid is 0.7-0.8mg with respect to 1L of the rooting medium.
In the present invention, in order to obtain sufficient illumination for tissue culture, culture conditions are set appropriately, and preferably, the culture conditions for the primary culture, the proliferation culture and the rooting culture are the same.
In the present invention, the culture conditions may be selected within a wide range, and preferably, the culture conditions are: the temperature is 22-28 ℃, the illumination time is 12-16h/d, and the illumination intensity is 1900-.
In the present invention, the time of the primary culture may be selected within a wide range, and preferably, the time of the primary culture is 30 days or more.
In the present invention, the time for the propagation culture can be selected within a wide range, and preferably, the time for the propagation culture is 30 days or more.
The present invention will be described in detail below by way of examples. In the following examples, the drugs and medicaments are all conventional commercial products.
Example 1
(1) Taking a tender branch of the current-year blueberry, and shearing a stem section with buds of 4 cm;
(2) disinfecting the stem with the buds for 30s by using 75% alcohol by volume concentration, then disinfecting for 10min by using 0.1% mercury bichloride by mass concentration, and flushing for later use;
(3) cutting the stem section with the buds into a single stem section with buds of 2cm, inserting the lower end of the single stem section into a WPM culture medium for primary culture, wherein the WPM culture medium contains 4mg of zeatin and 0.4mg of indolebutyric acid relative to 1L of the WPM culture medium;
(4) inoculating the single stem after primary culture into a WPM culture medium for propagation culture, wherein the content of zeatin and indolebutyric acid are 2mg and 0.2mg respectively relative to 1L of the WPM culture medium;
(5) inoculating the single stem after proliferation culture into 1/2WPM culture medium for rooting culture, wherein the content of indolebutyric acid is 0.8mg relative to 1L rooting culture medium.
Example 2
(1) Taking a tender branch of the current-year blueberry, and shearing a stem section with buds of 3 cm;
(2) disinfecting the stem with the buds for 20s by using alcohol with the volume concentration of 70%, disinfecting for 8min by using mercury bichloride with the mass concentration of 0.08%, and washing for later use;
(3) cutting the stem section with the buds into 1cm long single stem section with buds, inserting the lower end of the single stem section into a WPM culture medium for primary culture, wherein the WPM culture medium contains 3mg of zeatin and 0.3mg of indolebutyric acid relative to 1L of the WPM culture medium;
(4) inoculating the single stem after primary culture into a WPM culture medium for propagation culture, wherein the content of zeatin is 1mg and the content of indolebutyric acid is 0.1mg relative to 1L of the WPM culture medium;
(5) inoculating the single stem after proliferation culture into 1/2WPM culture medium for rooting culture, wherein the content of indolebutyric acid is 0.7mg relative to 1L rooting culture medium.
Example 3
(1) Taking a tender branch of the current-year blueberry, and shearing a stem section with buds of 4 cm;
(2) disinfecting the stem with the buds for 40s by using 75% alcohol by volume concentration, then disinfecting for 10min by using 0.12% mercury bichloride by mass concentration, and flushing for later use;
(3) cutting the stem section with the buds into single stem sections with buds of 2cm, inserting the lower ends of the single stem sections into a WPM culture medium for primary culture, wherein the WPM culture medium comprises 5mg of zeatin and 0.5mg of indolebutyric acid relative to 1L of the WPM culture medium;
(4) inoculating the single stem after primary culture into a WPM culture medium for propagation culture, wherein the content of zeatin and indolebutyric acid are 3mg and 0.3mg respectively relative to 1L of the WPM culture medium;
(5) inoculating the single stem after proliferation culture into 1/2WPM culture medium for rooting culture, wherein the content of indolebutyric acid is 0.9mg relative to 1L rooting culture medium.
Comparative example 1
(1) Taking a tender branch of the current-year blueberry, and shearing a stem section with buds of 3 cm;
(2) disinfecting the stem with the buds for 30s by using alcohol with the volume concentration of 75%, then disinfecting for 8min by using mercury bichloride with the mass concentration of 0.1%, and washing for later use;
(3) cutting the stem segment with the buds into single stem segments with the length of 1cm, inserting the lower ends of the single stem segments into an MS culture medium for primary culture, and relative to 1L of the MS culture medium, the single stem segments contain 4mg of zeatin and 0.4mg of indolebutyric acid;
(4) inoculating the single stem after primary culture into an MS culture medium for propagation culture, wherein the single stem contains 2mg of zeatin and 0.2mg of indolebutyric acid relative to 1L of MS culture medium;
(5) the single stem after proliferation culture is inoculated into 1/2MS culture medium for rooting culture, and relative to 1L 1/2MS culture medium, the content of indolebutyric acid is 0.8 mg.
Comparative example 2
(1) Taking a tender branch of the current-year blueberry, and shearing a stem section with buds of 4 cm;
(2) disinfecting the stem with the buds for 30s by using 75% alcohol by volume concentration, then disinfecting for 10min by using 0.1% mercury bichloride by mass concentration, and flushing for later use;
(3) cutting the stem segment with the buds into single stem segments with the length of 2cm, inserting the lower ends of the single stem segments into an MS culture medium for primary culture, and relative to 1L of the MS culture medium, the single stem segments contain 4mg of zeatin and 0.4mg of indolebutyric acid;
(4) inoculating the single stem after primary culture into an MS culture medium for propagation culture, wherein the single stem contains 2mg of zeatin and 0.2mg of indolebutyric acid relative to 1L of MS culture medium;
(5) the single stem after proliferation culture is inoculated into 1/2MS culture medium for rooting culture, and relative to 1L 1/2MS culture medium, the content of indolebutyric acid is 0.8 mg.
TABLE 1
| Example numbering | Growth of single-stem section after 30 days of propagation culture |
| Example 1 | Good growth vigor, low explant pollution rate and healthy growth of new leaves |
| Comparative example 1 | Poor growth vigor, high rate of contamination of the explants and sparse branches and leaves |
| Comparative example 2 | The growth vigor is general, the pollution rate of the explants is low, and the grown leaves are thin and small |
As can be seen from Table 1, the tissue culture seedlings of the "jewelry" blueberries grew well and appeared more branches and leaves after 30 days of propagation culture by using the method of example 1 in the scope of the present invention; the method of the comparative example 1 is used for culturing, and after 30 days of propagation culture, the growth vigor is poor, and the branches and leaves are sparse; after 30 days of proliferation, the culture is carried out by using the method of the comparative example 2, the growth vigor is general, the explant pollution rate is low, and the grown leaves are relatively thin. According to the method, the contents of hormones in the primary culture medium, the enrichment culture medium and the rooting culture medium are clearly and reasonably set, and reasonable sterilization treatment is carried out on the single stem section before inoculation, so that the tissue culture survival rate of the blueberries is high, and the tissue culture is easy to succeed.
The results of the culture methods of examples 2 to 3 substantially agreed with the results of the method of example 1.
The preferred embodiments of the present invention have been described in detail, however, the present invention is not limited to the specific details of the above embodiments, and various simple modifications may be made to the technical solution of the present invention within the technical idea of the present invention, and these simple modifications are within the protective scope of the present invention.
It should be noted that the various technical features described in the above embodiments can be combined in any suitable manner without contradiction, and the invention is not described in any way for the possible combinations in order to avoid unnecessary repetition.
In addition, any combination of the various embodiments of the present invention is also possible, and the same should be considered as the disclosure of the present invention as long as it does not depart from the spirit of the present invention.
Claims (10)
1. A method for culturing blueberry tissues, wherein the blueberry is a jewel, and the method comprises the following steps:
(1) selecting explants: taking the young branch of the current-year blueberry, and shearing the stem with the bud;
(2) and (3) disinfection treatment of explants: disinfecting the stem section with the buds by using alcohol, disinfecting the stem section with the bud by using mercury bichloride, and washing the stem section for later use to obtain a disinfected stem section with the buds;
(3) primary culture: cutting the sterilized stem section with buds into single stem sections with buds, and inserting the lower ends of the single stem sections into a primary culture medium for primary culture;
(4) and (3) proliferation culture: inoculating the single stem after primary culture into a proliferation culture medium for proliferation culture;
(5) rooting culture: inoculating the single stem section after propagation culture into a rooting culture medium for rooting culture;
the primary culture medium is a WPM culture medium, the proliferation culture medium is a WPM culture medium, and the rooting culture medium is 1/2WPM culture medium.
2. A method according to claim 1 wherein in step (1) the budded stem segment is 3-4cm in length.
3. The method of claim 1, wherein in step (2), the alcohol sterilization comprises: soaking the stem segments with the buds in alcohol with the volume fraction of 70-75%, and sterilizing for 20-40s by shaking;
preferably, the mercuric chloride disinfection is: soaking the stem segments with buds sterilized by alcohol into mercuric chloride with the mass fraction of 0.08-0.12%, and sterilizing for 8-10min by shaking;
more preferably, the washing adopts sterile washing for 5-6 times, and the time of each washing is 1-3 min.
4. The method according to claim 1, wherein, in step (3), the inoculation of the primary medium is performed around an alcohol lamp.
5. The method according to claim 1, wherein, in step (3), the single stem segment has a length of 1-2 cm;
preferably, the primary culture medium also contains zeatin and indolebutyric acid;
more preferably, the zeatin content is 3-5mg and the indolebutyric acid content is 0.3-0.5mg relative to 1L of the primary medium.
6. The method of claim 1, wherein the step (3) further comprises: the single stem sections were blotted dry of surface moisture with sterile filter paper.
7. The method according to claim 1, wherein the proliferation medium in step (4) further comprises zeatin and indolebutyric acid;
preferably, the content of zeatin is 1-3mg and the content of indolebutyric acid is 0.1-0.3mg relative to 1L of the proliferation medium.
8. The method according to claim 1, wherein, in step (5), the rooting medium further comprises indolebutyric acid;
preferably, the content of indolebutyric acid is 0.7-0.9mg with respect to 1L of the rooting medium.
9. The method of claim 1, wherein the primary culture, the propagation culture and the rooting culture are under the same culture conditions;
preferably, the culture conditions are: the temperature is 22-28 ℃, the illumination time is 12-16h/d, and the illumination intensity is 1900-.
10. The method according to claim 1, wherein the primary culture is carried out for 30 days or more;
preferably, the time for the propagation culture is 30 days or more.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN115918535A (en) * | 2022-12-02 | 2023-04-07 | 浙江省农业科学院 | Sugar-free blueberry tissue culture method |
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