A method for improving the success rate of regenerated plants of Curcuma alismatifolia

Abstract

The present invention discloses a method for improving the success rate of regenerated plants of Curcuma alismatifolia, belonging to the technical field of plant tissue culture. The present invention uses the latent buds on the underground tubers of Curcuma alismatifolia as the starting explants for in vitro tissue culture, which can be induced into complete regenerated plants, significantly reducing the contamination rate in the explant inoculation step. After proliferation, rooting and transplanting into seedlings, a large number of Curcuma alismatifolia seedlings with high phenotypic consistency and good genetic stability can be obtained.

Description

Technical Field

[0001] The present invention relates to the technical field of plant tissue culture, and particularly relates to a method for improving the success rate of regenerated plants of Curcuma alismatifolia. Background Art

[0002] There are many mutant varieties of Curcuma alismatifolia, and the chromosome ploidy within the genus is generally inconsistent. The efficiency of conventional cross-breeding methods is low. New varieties of Curcuma alismatifolia mainly rely on mutagenesis breeding. After screening individuals with excellent traits, asexual propagation is carried out to retain their excellent traits. The main method of asexual propagation of Curcuma alismatifolia is tissue culture. At present, the main explants for tissue culture of Curcuma alismatifolia are small balls, shoot tips or corms. However, these explants are large in volume, loose in texture, and directly contact the soil during cultivation. Therefore, the tissue is rich in endophytic bacteria. Although the induction rate is high, there are problems of high contamination rate and low success rate, and it is difficult to successfully propagate with a small starting amount in most cases. During the cultivation process of new varieties of Curcuma alismatifolia, the number of excellent individuals obtained by field screening is often small, and the low success rate of tissue culture is extremely unfavorable for the rapid propagation of excellent individuals. Therefore, it is particularly important to improve the success rate during the regeneration of Curcuma alismatifolia, which can meet the demand for cloning a large number of plants by asexual reproduction during the breeding process. Summary of the Invention

[0003] The purpose of the present invention is to provide a method for improving the success rate of regenerated plants of Curcuma alismatifolia to solve the problems existing in the above-mentioned prior art. The present invention uses the dormant buds on the underground tubers of Curcuma alismatifolia as the starting explants for in vitro tissue culture, which can be induced into complete regenerated plants, significantly reducing the contamination rate in the explant inoculation step. After proliferation, rooting and transplanting into seedlings, a large number of Curcuma alismatifolia seedlings with high phenotypic consistency and good genetic stability can be obtained.

[0004] To achieve the above purpose, the present invention provides the following scheme:

[0005] The present invention provides a method for improving the success rate of regenerated plants of Curcuma alismatifolia, comprising the following steps:

[0006] (1) Select a Curcuma alismatifolia plant with plump rhizomes and not reaching the flowering stage, cut open the part connecting the leaf base and the rhizome, and expose the dormant buds remaining in the leaf base;

[0007] In the present invention, the Curcuma alismatifolia plant is a plant in good growth condition and not reaching the flowering stage; in some embodiments of the present invention, the Curcuma alismatifolia plant is obtained by cultivating dormant corms for more than 2 months;

[0008] (2) Cut the dormant buds remaining in the leaf base, and only retain the tissue 2-3 mm around the dormant buds and the phloem on the surface of the rhizome to obtain explants;

[0009] (3) After disinfecting the explant, inoculate it into the adventitious bud induction medium and induce adventitious buds through culture.

[0010] (4) Transfer the adventitious buds to the proliferation medium and obtain clustered buds through proliferation culture.

[0011] (5) Transfer the clustered buds to a new proliferation medium and obtain tissue culture seedlings through subculture. After acclimatization, obtain the regenerated plants of Curcuma alismatifolia.

[0012] The proliferation medium is: MS medium + 5.8 g / L carrageenan + 30 g / L sucrose + 5 mg / L 6-BA + 0.5 mg / L NAA, pH = 5.8.

[0013] Preferably, the length of the explant is 6 - 8 mm.

[0014] Preferably, the disinfection process includes rinsing with running water, disinfecting with sodium hypochlorite solution, disinfecting with ethanol solution, and disinfecting with mercuric chloride solution.

[0015] In the present invention, the time for rinsing with running water is 2 - 3 hours.

[0016] In the present invention, the mass concentration of the sodium hypochlorite solution is 10%, and the disinfection time is 1 min.

[0017] In the present invention, the volume concentration of the ethanol solution is 70%, and the disinfection time is 1 min.

[0018] In the present invention, the volume concentration of the mercuric chloride solution is 1‰, and the disinfection time is 7 min.

[0019] Preferably, after disinfecting with the sodium hypochlorite solution, after disinfecting with the ethanol solution, and after disinfecting with the mercuric chloride solution, rinse with sterile water to facilitate the elution of the sodium hypochlorite solution, ethanol solution, and mercuric chloride solution.

[0020] In the present invention, after disinfecting with the sodium hypochlorite solution, rinse with sterile water once.

[0021] In the present invention, after disinfecting with the ethanol solution, rinse with sterile water once.

[0022] In the present invention, after disinfecting with the mercuric chloride solution, rinse with sterile water 5 times.

[0023] In the present invention, after disinfecting the explant and before inoculation, appropriately remove the browning tissue.

[0024] Preferably, the adventitious bud induction medium is: MS medium + 5 g / L carrageenan, pH = 5.8.

[0025] Preferably, the conditions for induction culture are: 24°C ± 2°C, dark culture; the time is 3 - 5 days.

[0026] Preferably, the conditions for proliferation culture are: 24°C ± 2°C, illumination for 10 ± 0.5 h / d, light intensity of 3000 - 5000 LX; the time is 30 - 40 days.

[0027] Preferably, the conditions for subculture are: 24°C ± 2°C, illumination for 10 ± 0.5 h / d, light intensity of 3000 - 5000 LX.

[0028] Preferably, the specifications of the tissue culture seedlings after subculture are 7 - 10 cm in length and 0.7 - 1 cm in basal stem width.

[0029] In the present invention, the acclimatization of seedlings adopts a conventional acclimatization management method; in some embodiments of the present invention, the culture medium during acclimatization is peat, the culture humidity is 90%, and the culture time is 1 - 2 weeks.

[0030] The present invention discloses the following technical effects:

[0031] In the present invention, the latent buds existing in the leaf base of the plant before the flowering stage of Curcuma alismatifolia are selected as the starting explants. The tissue composition mainly includes the phloem on the surface of the nutrient roots and the latent buds. The explants are small in size and high in activity, and can be induced to form Curcuma alismatifolia tissue culture seedlings, significantly reducing the contamination rate during the inoculation and induction of explants. The contamination rate within two weeks is only 8.6%, greatly improving the success rate of establishing tissue culture seedlings from Curcuma alismatifolia explants. The adventitious buds of Curcuma alismatifolia are subcultured and proliferated on the proliferation medium, and the proliferation coefficient reaches more than 4.7. The growth rate is fast, the growth trend is strong, and there is no need for rooting culture. After removing the medium, it can be planted and acclimatized. The culture process is simple, and a large number of regenerated plants with high phenotypic consistency and good genetic stability of Curcuma alismatifolia seedlings can be obtained. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required in the embodiments. Obviously, the drawings in the following description are only some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can be obtained based on these drawings.

[0033] Figure 1 The strip roots excised from the plant before flowering, the retained tubers, and the excised leaves in Example 1;

[0034] Figure 2 The leaf base on the rhizome of the plant before flowering in Example 1, and the latent buds are indicated by the red dotted line;

[0035] Figure 3The latent bud explants are cut from the plants before flowering in Example 1;

[0036] Figure 4 The latent buds and adventitious buds successfully induced in the plants before flowering in Example 1;

[0037] Figure 5 These are the ginger lotus clustered buds obtained by multiplication and subculture in Example 2. DETAILED DESCRIPTION

[0038] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0039] It should be understood that the terms described in the present invention are only for describing a particular embodiment and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. The intermediate value in any stated value or stated range, and each smaller range between any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0040] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0041] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to those skilled in the art. The present invention description and examples are exemplary only.

[0042] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0043] The variety of ginger lotus plants used as explants in the present invention is not limited. In some embodiments, the variety of ginger lotus plants is selected from Chiang Mai Pink, Red Guanyin, Dutch Red, Chiang Mai White, Snow White, etc.

[0044] Example 1

[0045] The contamination rate of the field ginger lotus plant tissue ('Chiang Mai Powder' variety) as explants, under the same sterilization process conditions, mainly depends on the degree of microbial invasion into the plant tissue and the degree of microbial killing ability of the allelopathic substances secreted by the plant itself.

[0046] In this example, the same tissue of ginger lotus, the persistent latent buds and bud tips in the leaf base of the tuber, are selected as explants, and the only difference is the material collection period, as shown in Table 1. Taking the plant before flowering as an example, the preparation of the explant and the process of adventitious bud induction are as follows:

[0047] (1) Collect ginger lotus plants that have been planted for more than two months since dormancy and have not yet produced inflorescences, rinse off the soil, cut off the leaves and strip roots, and only keep the tubers ( Figure 1 ), scrape off the remaining leaf bases on the tuber and expose the hidden buds stored in the leaf bases ( Figure 2 , the red dotted line is the hidden bud), and cut it, leaving only about 3mm of tissue around the hidden bud, and cut off the stem tissue below the hidden bud, leaving only the phloem, to obtain the explant ( Figure 3 ).

[0048] (2) The explants were placed under running water and rinsed for 3 hours, then transferred to a tissue culture operating table, disinfected with 10% sodium hypochlorite solution for 1 min, rinsed once with sterile water, disinfected with 70% ethanol solution for 1 min, rinsed once with sterile water, disinfected with 1‰ mercuric chloride solution for 7 min, rinsed 5 times with sterile water, and placed on a sterile plate for use. After the browned marginal tissue was appropriately cut off, the explants were inoculated into adventitious bud induction medium (MS medium + carrageenan 5 g / L, pH = 5.8), and cultured at 25°C ± 1°C, light intensity 10 ± 0.5 h / d, and light intensity 4000 LX for 14 days to obtain adventitious buds ( Figure 4 ).

[0049] The number of adventitious buds contaminated by different explants was counted, the contamination rate was calculated, and the types of contaminating bacteria in the adventitious buds were detected. The results are shown in Table 1.

[0050] Table 1 Contamination rate of ginger lotus explants at different sampling periods

[0051] Explants Number of explants Number of contaminated explants Contamination rate Types of contaminating bacteria Latent buds of primary plants 134 128 95.5％ Actinomycetes, bacteria Shoot tips of primary plants 47 43 91.4％ Actinomycetes, bacteria, fungi Latent buds of pre-growth stage plants 122 92 75.4％ Actinomycetes, bacteria, fungi Shoot tips of pre-growth stage plants 51 39 76.5％ Actinomycetes, bacteria, fungi Latent buds of pre-flowering plants 163 14 8.6％ Fungi Shoot tips of pre-flowering plants 63 6 9.5％ Fungi

[0052] Table 1 shows that the contamination rate of explants decreased significantly with the growth period of the sampled plants; the contamination rate in the primary plants after the ginger lotus bulbs were dormant was as high as 95.5%, and almost no usable materials were obtained. The contamination rate of the same explant site before flowering was reduced to 8.5%, and more usable materials were obtained with good activity and high adventitious bud induction rate, which significantly improved the success rate of tissue culture seedling establishment.

[0053] Example 2

[0054] In this example, the adventitious buds induced from the rhizome leaf base buds before flower extraction in Example 1 were used as materials, and were subjected to proliferation culture in a proliferation medium supplemented with different plant growth regulators. After obtaining clustered buds ( Figure 5 ), the clustered buds were transferred to a new proliferation medium for subculture. The formula of the proliferation medium is shown in Table 2. 60 adventitious buds were used in each treatment group, and all were cultured at 25°C ± 1°C, with a light intensity of 4000 LX and a light period of 10 ± 0.5 h / d. After 30 days of proliferation culture and 90 days of subculture, the number of proliferated buds was counted and the proliferation coefficient was calculated. The proliferation coefficient = (number of induced new buds / number of inoculated adventitious buds) × 100%, and the growth trend of the tissue culture seedlings was observed. The results are shown in Table 2.

[0055] Table 2 Proliferation coefficients of proliferation media with different formulations

[0056]

[0057] Table 2 shows that taking the ordinary MS medium as the control, the proliferation coefficient was the highest in Medium No. 3 (MS + 5.8 g / L carrageenan + 30 g / L sucrose + 5 mg / L 6-BA + 0.5 mg / L NAA) containing plant growth regulators, which was 4.77, significantly higher than that of the control group and the media containing low concentrations of 6-BA and equal concentrations of TDZ. The tissue culture seedlings proliferated and subcultured in Medium No. 3 had strong growth and vigorous and thick roots. Therefore, Medium No. 3 was selected for proliferation culture.

[0058] The above-described examples are only descriptions of the preferred embodiments of the present invention, and do not limit the scope of the present invention. Without departing from the design spirit of the present invention, various deformations and improvements made by those of ordinary skill in the art to the technical solutions of the present invention shall fall within the protection scope determined by the claims of the present invention.

Claims

1. A method for improving the success rate of ginger lotus plant regeneration, characterized in that: The steps include: (1) Select ginger lotus plants with full rhizomes and before flowering, cut the part where the leaf base is connected to the rhizome, and expose the hidden buds at the leaf base; (2) cutting the hidden buds stored at the leaf base, retaining only 2-3 mm of tissue around the hidden buds and the phloem on the surface of the rhizome, to obtain explants; (3) sterilizing the explants, inoculating them into an adventitious bud induction medium, and inducing them to obtain adventitious buds; (4) transferring the adventitious buds to a proliferation medium and performing proliferation culture to obtain clustered buds; (5) transferring the clustered buds to a new proliferation medium, subculturing to obtain tissue culture seedlings, and hardening the seedlings to obtain ginger lotus regenerated plants; The adventitious bud induction medium is: MS medium + 5g / L carrageenan, pH = 5.8; The proliferation culture medium is: MS culture medium + 5.8 g / L carrageenan + 30 g / L sucrose + 5 mg / L 6-BA + 0.5 mg / L NAA, pH = 5.

8.

2. The method according to claim 1, characterized in that The length of the explant is 6-8 mm.

3. The method according to claim 1, characterized in that The disinfection process includes running water flushing, sodium hypochlorite solution disinfection, ethanol solution disinfection, and mercuric chloride solution disinfection.

4. The method according to claim 3, characterized in that After the disinfection with the sodium hypochlorite solution, the disinfection with the ethanol solution, and the disinfection with the mercuric chloride solution, sterile water is used for rinsing.

5. The method according to claim 1, characterized in that The conditions of the proliferation culture are: 24°C±2°C, light intensity of 10±0.5h / d, light intensity of 3000-5000LX; and time of 30-40d.

6. The method according to claim 1, characterized in that The conditions of the subculture are: 24°C±2°C, light intensity of 10±0.5h / d, and light intensity of 3000-5000LX.

7. The method according to claim 1, characterized in that The specifications of the subculture to tissue culture seedlings are 7-10 cm long and 0.7-1 cm wide at the base of the stem.

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