Induced culture method of jujube pulp callus

Abstract

The invention discloses an induced culture method of jujube pulp callus, and belongs to the technical field of pulp callus. The jujube pulp callus induction culture method provided by the invention comprises the following steps: (1) sterilizing and peeling jujube fruits to obtain jujube pulp; and (2) cutting the jujube pulp, inoculating the jujube pulp to a callus induction culture medium, and culturing to obtain the jujube pulp callus. According to the invention, the induction culture method of the 'Jing jujube 60' fruit callus is successfully established, the induction rate is as high as 100%, and the most basic test material is provided for molecular function verification, genetic transformation and the like related to jujube fruits.

Description

Technical Field

[0001] This invention relates to the field of fruit pulp callus technology, and in particular to a method for inducing and culturing jujube fruit pulp callus. Background Technology

[0002] Jujube (Ziziphus jujuba Mill.) is a plant belonging to the genus Ziziphus in the family Rhamnaceae. It is a distinctive fruit tree with abundant germplasm resources, a long history of cultivation, and strong resistance and adaptability, possessing high economic, ecological, and social benefits. With the large-scale cultivation of jujube trees, the jujube industry is showing broad prospects. Jujube fruit is sweet and nutritious, considered a "woody food" and a "food and medicine of the same origin," rich in flavonoids, alkaloids, saponins, and other active substances. It possesses various pharmacological effects such as anti-oxidation, antibacterial and anti-inflammatory properties, and prevention of cardiovascular and cerebrovascular diseases, and is widely used in the food, pharmaceutical, and cosmetic industries.

[0003] Efficient tissue culture systems are fundamental for various biotechnological applications, including cell engineering and genetic engineering. Callus refers to a mass of undifferentiated cells that forms after a part of a plant is damaged or cut. It proliferates on a suitable culture medium and can undergo active cell division. As a common experimental material, callus is widely used in research on plant molecular mechanisms and genetic engineering.

[0004] Current reports on plant tissue culture of jujube trees mainly focus on stem segments, immature embryos, leaves, and anthers. These systems are relatively stable and mature, and can be used for gene function research. With technological advancements, the molecular mechanisms related to the formation of jujube fruit quality traits have gradually become a research hotspot. In the study of jujube fruit molecular mechanisms, callus tissue is a commonly used experimental material. However, callus tissue from other organs is not suitable for studying genes related to jujube fruit quality formation. Therefore, fruit callus tissue has irreplaceable advantages compared to callus tissue from other organs. Cultured jujube pulp callus tissue is a very important approach to studying the molecular mechanisms related to jujube fruit quality formation.

[0005] Furthermore, by optimizing the conditions and induction methods in plant tissue culture, the formation efficiency and regeneration capacity of callus tissue can be improved, thereby establishing a relatively complete callus transformation system. This can provide a foundation for improving crop varieties, enhancing the quality and yield of jujube fruits through transgenic technology. Summary of the Invention

[0006] The purpose of this invention is to provide a method for inducing and culturing jujube pulp callus to solve the problems existing in the prior art. This invention provides a method for inducing and culturing jujube pulp callus and its proliferation, establishes an efficient and rapid jujube pulp callus culture system, improves the callus differentiation rate of jujube fruit, provides experimental materials for verifying the molecular mechanisms of jujube fruit quality, and provides methods and ideas for molecular biology and genetic engineering research on the formation of jujube fruit quality traits.

[0007] To achieve the above objectives, the present invention provides the following solution:

[0008] In a first aspect, the present invention provides a method for inducing and culturing jujube pulp callus, comprising the following steps:

[0009] (1) Disinfect and peel the jujube fruit to obtain jujube pulp;

[0010] (2) Cut the jujube pulp and inoculate it into the callus induction medium. Culture it at 23-27℃ for 25-35 days to obtain jujube pulp callus.

[0011] The callus induction medium consisted of: MS 4.0-5.0 g / L, sucrose 20-40 g / L, agar powder 6-10 g / L, TDZ 0.2-1.5 mg / L and NAA 0.2-1.0 mg / L, with a pH of 5.8-6.0.

[0012] Preferably, the jujube fruit variety is Jingzao 60.

[0013] Preferably, the jujube fruit is in the young fruit stage or the fruit enlargement stage.

[0014] Preferably, the disinfection method in step (1) is as follows: after rinsing the jujube fruit with water, disinfect it in sequence with 0.5-1.5% sodium hypochlorite and 70-80% alcohol.

[0015] Preferably, the jujube pulp is square after being cut.

[0016] Preferably, the culture temperature in step (2) is 25°C and the culture time is 30 days.

[0017] Preferably, the callus induction culture medium in step (2) consists of MS 4.5 g / L, sucrose 30 g / L, agar powder 8 g / L, TDZ 0.5 mg / L and NAA 0.5 mg / L.

[0018] Secondly, the present invention also provides the application of jujube pulp callus obtained by the aforementioned induction culture method as a material for jujube genetic transformation experiments.

[0019] The present invention discloses the following technical effects:

[0020] This invention induced and cultured callus tissue from 'Jingzao 60' jujube using 10 different hormone ratios induction media. By comparing the induction rate and callus growth, a suitable medium for efficient induction and proliferation of 'Jingzao 60' callus tissue was identified: MS 4.5 g / L + sucrose 30 g / L + agar powder 8 g / L + TDZ 0.5 mg / L + NAA 0.5 mg / L (pH 5.8-6.0). This provides fundamental experimental material for molecular functional verification and genetic transformation of jujube fruit. Attached Figure Description

[0021] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0022] Figure 1 The results represent callus induction; where A represents the young fruit stage of Jingzao 60, and B represents the fruit enlargement stage of Jingzao 60.

[0023] Figure 2 This represents the result of callus proliferation; among them, A and B are both young fruit stages of jujube 60. Detailed Implementation

[0024] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0025] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0026] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0027] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be readily apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0028] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0029] Example 1

[0030] 1. Experimental Materials

[0031] Fruits of 'Jingzao 60' during the young fruit stage (10-20 days after flowering) and the fruit enlargement stage (50 days after flowering).

[0032] 2. Experimental Methods

[0033] (1) Explant disinfection

[0034] Rinse the collected jujube fruits with running water for 25 minutes, turning them over continuously during this time. Wipe them dry with paper and set aside. In a clean bench, first disinfect with 1% sodium hypochlorite for 8 minutes, then disinfect with 75% alcohol for 10-15 minutes. Rinse the jujube fruits with sterile water 2-3 times, 2-3 minutes each time. Finally, wipe the water off the surface of the jujube fruits with sterile toilet paper.

[0035] (2) Induction and culture of callus

[0036] Using MS as the basal medium, 10 different ratios of induction media with different concentrations of hormones (NAA and TDZ) were selected, as shown in Table 1. The media were then autoclaved at 121℃ for 20 min.

[0037] Table 110 different induction culture media

[0038]

[0039]

[0040] After disinfection and cleaning, peel the jujube fruit with a sterilized knife, place the pulp on sterilized filter paper, and then cut the peeled jujube pulp into pieces of about 1cm×1cm×0.3cm. Spread them evenly on 10 different induction culture media and culture them in a dark environment at 25℃ for 30 days.

[0041] The callus induction medium composition is as follows (1L water): MS 4.5g / L + sucrose 30g / L + agar powder 8g / L + TDZ 0.2-1.5mg / L + NAA 0.2-1.0mg / L, pH=5.8-6.0.

[0042] (3) Results Statistics

[0043] Induction rate = (number of inoculated tissues that induce callus / total number of inoculated tissues) × 100%. The callus induction rate of different induction culture media is calculated according to the formula.

[0044] (4) Callus proliferation culture

[0045] The jujube pulp callus obtained after induction culture was inoculated onto the culture medium with the highest induction rate and the best effect selected above and cultured for a longer period of time.

[0046] 3. Test Results

[0047] from Figure 1 As shown in Table 2, the optimal induction medium for 'Jingzao 60' is: MS 4.5g / L + 30g / L sucrose + 8g / L agar powder + 0.5mg / L TDZ + 0.5mg / L NAA. Under this medium, the callus induction rate of 'Jingzao 60' fruit during the young fruit stage can reach 100%, and the callus induction rate of fruit during the fruit enlargement stage can reach 92%. The callus induction rate of 'Jingzao 60' jujube pulp is higher during the young fruit stage than during the fruit enlargement stage.

[0048] After proliferation culture on the same culture medium (cultured at 25℃ for 30-40 days), the callus tissue was yellowish-white, dense, and exhibited strong differentiation ability, as can be seen in the following figures. Figure 2 .

[0049] Table 2 Callus induction rate in different culture media

[0050]

[0051]

[0052] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A method for inducing and culturing jujube pulp callus, characterized in that, Includes the following steps: (1) Disinfect and peel the jujube fruit to obtain jujube pulp; (2) Cut the jujube pulp and inoculate it into the callus induction medium. Culture it at 23-27℃ for 25-35 days to obtain jujube pulp callus. The callus induction medium consisted of: MS 4.0-5.0 g / L, sucrose 20-40 g / L, agar powder 6-10 g / L, TDZ 0.2-1.5 mg / L and NAA 0.2-1.0 mg / L, with a pH of 5.8-6.

0.

2. The induction culture method according to claim 1, characterized in that, The jujube fruit variety mentioned is Jingzao 60.

3. The induction culture method according to claim 1, characterized in that, The jujube fruit in question is either in its young fruit stage or in its enlargement stage.

4. The induction culture method according to claim 1, characterized in that, The disinfection method in step (1) is as follows: after rinsing the jujube fruit with water, disinfect it in sequence with 0.5-1.5% sodium hypochlorite and 70-80% alcohol.

5. The induction culture method according to claim 1, characterized in that, The jujube pulp is square after being cut.

6. The induction culture method according to claim 1, characterized in that, The culture temperature in step (2) is 25℃ and the culture time is 30 days.

7. The induction culture method according to claim 1, characterized in that, The callus induction culture medium in step (2) consists of MS 4.5 g / L, sucrose 30 g / L, agar powder 8 g / L, TDZ 0.5 mg / L and NAA 0.5 mg / L.

8. The application of jujube pulp callus obtained by the induction culture method according to any one of claims 1-7 as a material for jujube genetic transformation experiments.

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