Method for efficiently inducing muskmelon callus, culture medium composition and application

Abstract

The invention discloses a method for efficiently inducing muskmelon calluses, a culture medium composition and application, and belongs to the technical field of plant tissue culture. The method comprises the following steps: selecting melon cotyledons at the seedling age of 5 days as explants, and inoculating the explants into a callus induction culture medium for induction culture to form calluses; the obtained callus is transferred to an adventitious bud differentiation culture medium for differentiation culture, adventitious buds are obtained, cotyledon explants in a specific development state (the seedling age of 5 days) are selected and matched with optimized hormone combinations (such as 1.0 mg / L 6-BA and 0.1 mg / L IAA), the induction rate and quality of the callus are remarkably improved, the callus is loose in structure and high in embryogenic property, and the survival rate of the adventitious buds is increased. And a high-quality raw material is provided for subsequent genetic operation.

Description

Technical Field

[0001] This invention relates to the field of plant tissue culture technology, and in particular to a method, culture medium composition and application for efficiently inducing callus tissue from melon. Background Technology

[0002] Muskmelon (Cucumis melo L.) is an important horticultural economic crop, prized for its delicious and nutritious fruit, which enjoys strong market demand. With the development of modern breeding techniques, tissue culture has become a key technology for muskmelon variety improvement and germplasm innovation. Establishing an efficient and stable plant regeneration system is a prerequisite for conducting molecular breeding work such as gene editing and genetic transformation. In this system, callus induction and culture are fundamental steps. Callus, as a dedifferentiated cell mass, possesses the totipotency to regenerate complete plants and can be considered the "raw material" and "processing plant" for genetic manipulation. Its induction efficiency and quality directly affect subsequent gene introduction, plant regeneration, and the breeding cycle.

[0003] Currently, callus induction in melon tissue culture still faces many technical bottlenecks. First, different melon varieties, especially thick-skinned types, exhibit significant differences in their response to culture media and hormone formulations, generally exhibiting low callus induction rates, severe browning, and slow growth, making it difficult to obtain large quantities of embryogenic callus suitable for genetic manipulation. Second, existing induction systems are mostly optimized for specific varieties, lacking versatility and difficult to directly apply to other superior germplasm, thus limiting the widespread adoption of gene breeding technology. Furthermore, traditional methods often rely on complex combinations of exogenous hormones and culture conditions, involving cumbersome procedures with low reproducibility, hindering efficient and large-scale production. The root causes of these problems lie in insufficient understanding of the developmental state of melon cotyledon explants, inadequate optimization of hormone types and concentration ratios, and imprecise control of the culture environment, leading to incomplete cell dedifferentiation and inconsistent callus quality.

[0004] Therefore, developing a tissue culture method applicable to multiple melon varieties, simple to operate, with high induction rate and excellent callus quality is of great significance for breaking through the bottleneck of melon genetic transformation and accelerating the creation of superior germplasm. However, existing technologies still have significant shortcomings in achieving efficient induction and stable regeneration. There is an urgent need for systematic optimization of key aspects such as explant selection, culture medium formulation, and culture conditions to establish a reproducible and easily scalable efficient induction system, providing reliable technical support for melon molecular breeding. Summary of the Invention

[0005] The purpose of this invention is to provide a method, culture medium composition, and application for efficiently inducing callus tissue in melon, thereby addressing the problems existing in the prior art. This invention significantly improves the callus induction rate and quality in melon by optimizing explant state and hormone ratios, and constructs a complete regeneration system. This method is simple to operate, has good reproducibility, and provides an efficient and reliable technical platform for melon genetic transformation and breeding.

[0006] To achieve the above objectives, the present invention provides the following solution:

[0007] This invention provides a method for efficiently inducing callus tissue in melon, comprising the following steps:

[0008] Five-day-old melon seedlings were selected as explants and inoculated into callus induction medium to induce callus formation.

[0009] The obtained callus tissue was transferred to an adventitious shoot differentiation medium for differentiation culture to obtain adventitious shoots;

[0010] The callus induction medium comprises MS basal medium + 1.0 mg / L 6-BA + 0.1 mg / L IAA, or MS basal medium + 0.5 mg / L 6-BA + 0.5 mg / L IAA, or MS basal medium + 1.5 mg / L 6-BA + 0.1 mg / L IAA, or MS basal medium + 1.5 mg / L 6-BA + 0.3 mg / L IAA;

[0011] The components of the adventitious bud differentiation medium include MS basal medium + 1.0 mg / L 6-BA + 0.1 mg / L IAA.

[0012] Optionally, the induction culture conditions are: temperature 25±2℃, light intensity 1500-2000 lx, photoperiod 16h, and culture for 15-20 days.

[0013] Optionally, the differentiation culture conditions are a temperature of 25±2℃, a photoperiod of 16h, and a culture period of 15 days.

[0014] Optionally, after obtaining the adventitious buds, the method further includes a step of adventitious bud elongation growth; the step of adventitious bud elongation growth includes transferring the adventitious buds to an elongation culture medium for cultivation to promote the elongation growth of the adventitious buds.

[0015] The elongation medium consists of MS basal medium + 0.05 mg / L 6-BA + 0.15 mg / L GA3.

[0016] Optionally, the culture conditions are a temperature of 25±2℃, a photoperiod of 16h, and a culture period of 15-20 days.

[0017] Optionally, after the adventitious buds elongate, the process may also include the induction of adventitious root growth; the induction of adventitious root growth includes transferring the elongated adventitious buds to a rooting medium to induce the generation of adventitious roots.

[0018] The rooting medium consists of MS basal medium + 0.8 mg / L IAA.

[0019] Optionally, the conditions for inducing adventitious root formation are a temperature of 25±2℃, a photoperiod of 16h, and a culture period of 15 days.

[0020] The present invention also provides a culture medium composition for efficiently inducing callus in melon, comprising a callus induction medium and an adventitious shoot differentiation medium;

[0021] The callus induction medium comprises MS basal medium + 1.0 mg / L 6-BA + 0.1 mg / L IAA, or MS basal medium + 0.5 mg / L 6-BA + 0.5 mg / L IAA, or MS basal medium + 1.5 mg / L 6-BA + 0.1 mg / L IAA, or MS basal medium + 1.5 mg / L 6-BA + 0.3 mg / L IAA;

[0022] The components of the adventitious bud differentiation medium include MS basal medium + 1.0 mg / L 6-BA + 0.1 mg / L IAA.

[0023] Optionally, it may also include at least one of adventitious shoot elongation medium and rooting medium;

[0024] The adventitious shoot elongation medium consists of MS basal medium + 0.05 mg / L 6-BA + 0.15 mg / L GA3;

[0025] The rooting medium consists of MS basal medium + 0.8 mg / L IAA.

[0026] The present invention also provides the application of the culture medium composition described above in improving the callus formation and development ability of melon.

[0027] The present invention discloses the following technical effects:

[0028] The method and accompanying culture medium composition for efficiently inducing callus in melon provided by this invention effectively solve key problems in existing technologies, such as low callus induction rate, unstable system, and poor variety applicability. By selecting cotyledon explants at a specific developmental stage (5-day seedling age) and combining them with an optimized hormone combination, the induction rate and quality of callus are significantly improved, resulting in a loose structure and strong embryogenicity, providing high-quality raw materials for subsequent genetic operations.

[0029] This method further establishes a complete regeneration system from callus induction, adventitious bud differentiation, elongation growth to rooting and seedling formation. The culture medium formulations for each step are clearly defined, the culture conditions are controllable, and the operation is simple and reproducible. Experiments show that this system exhibits good induction effects on multiple thick-skinned melon varieties, and is particularly suitable for superior germplasm such as '18J-3-9', providing a reliable and efficient technical platform for achieving efficient genetic transformation, germplasm innovation, and large-scale seedling production in melons. Attached Figure Description

[0030] 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.

[0031] Figure 1 This is a diagram illustrating the division of melon cotyledons; where A represents melon cotyledons at 5 days of seedling age; and B and C represent different tissue parts of the divided melon cotyledons.

[0032] Figure 2 This study compares the growth status of melon callus tissue on different culture media; where NO.1 represents '156'; NO.2 represents '18J-3-9'; and M1-M10 represent 10 different hormone ratios in different culture media.

[0033] Figure 3 Comparative images of the adventitious bud elongation growth of two melon varieties; where No. 1 is '156' and No. 2 is '18J-3-9'.

[0034] Figure 4 These represent different stages in the formation process of melon regenerated plants; where A is the callus stage; B is the bud differentiation stage; C is the bud growth stage; D is the root differentiation stage; E is the hardening-off stage; and F is the regenerated plant stage. Detailed Implementation

[0035] 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.

[0036] 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.

[0037] 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.

[0038] 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 apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0039] 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.

[0040] The terminology used in this invention:

[0041] 6-BA is 6-benzyladenine; IAA is 3-indoleacetic acid; GA3 is gibberellin; the reagents used in the embodiments of this invention are all commercially available.

[0042] In this invention, the melon varieties '156' and '18J-3-9' selected as explants are disclosed in the literature "Analysis of changes in expression of carotenoid metabolism-related genes, sugar accumulation and metabolism-related enzyme activity in different melon inbred lines, Shanghai Journal of Agricultural Sciences, March 30, 2021" and "Effects of downy mildew on physiological and biochemical indicators of different resistant melons, Shanghai Vegetables, February 15, 2025".

[0043] Example

[0044] 1. Selection and sterilization of explants

[0045] One hundred plump seeds each of the thick-skinned melon varieties '156' and '18J-3-9' were selected. After removing the shells, the seeds were sterilized by soaking in 75% alcohol for 30 seconds, followed by sterilization in a 2% sodium hypochlorite solution for 15 minutes. The seeds were then rinsed 3-4 times with sterile water for 1 minute each time in a laminar flow hood. The surface-sterilized seeds were inoculated into MS basal medium for seed embryo germination. After inoculation, the seeds were incubated in the dark at 25±2 ℃ for 2 days, followed by photoperiodic culture at a light intensity of 1500-2000 lx for 16 hours daily until day 5.

[0046] 2. Preparation of explants

[0047] Take the above-mentioned melon seedlings that have been cultured for 5 days. At this time, the cotyledons are tightly closed and have a high meristematic capacity. Under aseptic conditions, remove the growing point, adaxial end, and abaxial end of the seedling, retaining two intact cotyledons. Make a longitudinal cut along the midrib of each cotyledon, and then a transverse cut, dividing each cotyledon into 4 equal pieces. Therefore, 8 explants can be obtained from each seed. Figure 1 The cotyledon cells at this stage have a strong ability to differentiate, which is beneficial for the subsequent induction of callus tissue.

[0048] 3. Callus induction

[0049] The segmented explants were inoculated into callus induction medium.

[0050] The formula for callus induction medium was: MS basal medium + 1.0 mg / L 6-BA + 0.1 mg / L IAA + 30 mg / L sucrose. 12-14 explants were inoculated into each 90 mm diameter, 20 mm high culture dish. Culture conditions were: temperature 25±2℃, light intensity 1500-2000 lx, 16 h light per day, for 15-20 days, at which point callus formation began at the explant incision site. The callus induction rate was recorded. During the culture period, the induction medium was replaced with fresh medium every 10 days.

[0051] This invention tested the induction effects of 10 different hormone ratios on callus induction media for melon. Specific hormone ratios are shown in Tables 1 and 2. A comparison of the growth status of callus from different melon varieties on different culture media is also provided. Figure 2 As shown.

[0052] Continuous observation revealed differences in the effectiveness of different treatments in inducing callus formation, with buds differentiating after approximately 25 days of induction. According to Table 1, for material 1 ('156'), except for the hormone ratios in M4, M8, and M10, the other combinations failed to completely induce callus formation. Specifically, the bud induction rate was higher when the 6-BA concentration was 1.0 mg / L and the IAA concentration was 0.3 mg / L, and when the 6-BA concentration was 1.5 mg / L and the IAA concentration was 0.1 mg / L or 0.3 mg / L. According to Table 2, for material 2 ('18J-3-9'), except for the hormone ratios in M1 and M10, all other combinations induced 100% callus formation. The bud induction rate reached its highest when the 6-BA concentration was 1.0 mg / L and the IAA concentration was 0.1 mg / L. Although the bud induction rate of bud 1 ('156') was higher than that of bud 2 ('18J-3-9'), the buds of bud 2 ('18J-3-9') showed better growth. Figure 3 It is clearly evident that the adventitious shoots of strain 2 ('18J-3-9') are longer and more robust than those of strain 1 ('156'), and its callus induction rate is also higher than that of strain 1 ('156'). In conclusion, strain 2 ('18J-3-9') can serve as a high-quality chassis resource for genetic transformation.

[0053] Table 1. Different concentration combinations of IAA and 6-BA and their induction effects in No. 1 ('156').

[0054]

[0055] Table 2. Different concentration combinations of IAA and 6-BA and their induction effects in No. 2 ('18J-3-9').

[0056]

[0057] 4. Adventitious bud differentiation

[0058] The induced loosely structured embryonic callus (such as...) Figure 4 (As shown in A) was transferred to differentiation medium. The differentiation medium formula was: MS basal medium + 1.0 mg / L 6-BA + 0.1 mg / L IAA. 6-9 callus tissues were inoculated into each culture dish, using plastic culture dishes with a diameter of 90 mm and a height of 20 mm. The culture conditions were set as follows: temperature 25 ± 2℃, photoperiod 16 h. Buds differentiated after approximately 10 days of culture, and adventitious buds were obtained after 15 days. The culture medium was replaced with fresh medium every 10 days during the culture period. Figure 4 B).

[0059] 5. Adventitious bud elongation growth

[0060] Select robust adventitious buds from the differentiation medium, cut them off from their base, and inoculate them into the adventitious bud elongation medium.

[0061] The elongation medium was formulated as follows: MS basal medium + 0.05 mg / L 6-BA + 0.15 mg / L GA3. After culturing at 25±2℃ and a 16-hour photoperiod for 15-20 days, adventitious buds elongated to approximately 3 cm and developed 5-6 true leaves, resulting in robust plants. Figure 4 (C).

[0062] 6. Adventitious root induction (rooting)

[0063] Cut off the well-grown adventitious buds from the base, remove the excess callus tissue attached to the base, and inoculate them into the rooting medium.

[0064] The rooting medium was formulated as follows: MS basal medium + 0.8 mg / L IAA. After culturing at 25±2℃ and a 16-hour photoperiod for approximately 15 days, a moderate number of robust adventitious roots were induced from the base of the adventitious buds. Figure 4 (D), forming a complete regenerated tissue culture seedling.

[0065] 7. Hardening off seedlings and transplanting

[0066] Move the rooted tissue culture seedlings, along with their culture bottles, to an outdoor environment or one with natural light. Open the bottle caps to harden the seedlings for 8-10 days, allowing them to gradually adapt to the external environment. After hardening, carefully remove the seedlings and gently wash away any plant gel adhering to the roots with clean water. Transplant the seedlings into a sterilized mixed substrate (…). Figure 4 The mixed substrate (E) is composed of soil and vermiculite mixed in a 1:1 volume ratio. After cultivating in the mixed substrate for approximately 20 days, robust plants are obtained and can then be transplanted to a greenhouse as regenerated plants. Figure 4 (F).

[0067] 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 efficient induction of melon callus, characterized by, The method comprises the following steps: The cotyledon of the melon at the age of 5 days is selected as the explant, and is inoculated into a callus induction medium to induce culture to form callus; The obtained callus is transferred into an adventitious bud differentiation medium to perform differentiation culture, and the adventitious bud is obtained; The component of the callus induction medium comprises MS basic medium+1.0 mg / L 6-BA+0.1 mg / L IAA, or MS basic medium+0.5 mg / L 6-BA+0.5 mg / L IAA, or MS basic medium+1.5 mg / L 6-BA+0.1 mg / L IAA, or MS basic medium+1.5 mg / L 6-BA+0.3 mg / L IAA; The component of the adventitious bud differentiation medium comprises MS basic medium+1.0 mg / L 6-BA+0.1 mg / L IAA.

2. The method of claim 1, wherein, The induction culture is performed at a temperature of 25±2℃, an illumination intensity of 1500-2000 lx, and a light cycle of 16h, and is cultured for 15-20 days.

3. The method of claim 1, wherein, The differentiation culture is performed at a temperature of 25±2℃ and a light cycle of 16h, and is cultured for 15 days.

4. The method of claim 1, wherein, After the adventitious bud is obtained, a step of elongation growth of the adventitious bud is further included; the step of elongation growth of the adventitious bud comprises transferring the adventitious bud into an elongation medium to perform culture, and promoting the elongation growth of the adventitious bud; The component of the elongation medium comprises MS basic medium+0.05 mg / L 6-BA+0.15 mg / L GA3.

5. The method of claim 4, wherein, The culture is performed at a temperature of 25±2℃ and a light cycle of 16h, and is cultured for 15-20 days.

6. The method of claim 4, wherein, After the adventitious bud is elongated, a step of inducing growth of adventitious roots is further included; the step of inducing growth of adventitious roots comprises transferring the adventitious bud after the elongation growth into a rooting medium to induce generation of the adventitious roots; The component of the rooting medium comprises MS basic medium+0.8 mg / L IAA.

7. The method of claim 6, wherein, The condition of inducing the generation of the adventitious roots is a temperature of 25±2℃, a light cycle of 16h, and culture for 15 days.

8. A medium composition for efficient induction of melon callus, characterized by, The callus induction medium and the adventitious bud differentiation medium are included; The component of the callus induction medium comprises MS basic medium+1.0 mg / L 6-BA+0.1 mg / L IAA, or MS basic medium+0.5 mg / L 6-BA+0.5 mg / L IAA, or MS basic medium+1.5 mg / L 6-BA+0.1 mg / L IAA, or MS basic medium+1.5 mg / L 6-BA+0.3 mg / L IAA; The component of the adventitious bud differentiation medium comprises MS basic medium+1.0 mg / L 6-BA+0.1 mg / L IAA.

9. The medium composition of claim 8, wherein, At least one of the adventitious bud elongation medium and the rooting medium is further included; The component of the adventitious bud elongation medium comprises MS basic medium+0.05 mg / L 6-BA+0.15 mg / L GA3; The component of the rooting medium comprises MS basic medium+0.8 mg / L IAA.

10. Use of the medium composition of claim 8 or 9 for increasing the ability of melon callus formation and development.

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