Callus Induction, Proliferation, and Differentiation Media for Longan Anthers and Their Applications

Abstract

The present invention belongs to the technical field of plant culture, and specifically relates to a callus induction, proliferation, and differentiation medium for longan anther culture and its application. The present invention includes 5 technical links: disinfection treatment of longan flower buds, explant extraction, anther callus induction, callus subculture proliferation, and callus differentiation; the method uses flower buds at the late uninucleate stage as materials, disinfects them with 75% alcohol for 10 s to 60 s, rinses them several times with sterile water and then soaks them in a little sterile water. This method can control the browning and contamination rate of explants within 20%; by adding activated carbon to the medium to adsorb harmful substances and inhibit the decomposition of phenolic substances, the callus induction rate is guaranteed to be above 80%; and the callus is proliferated, and a large amount of callus can be obtained in 2 to 3 months.

Description

Technical Field

[0001] The present invention belongs to the technical field of plant cultivation, and particularly relates to a callus induction, proliferation, and differentiation medium for longan anther culture and its application. Background Art

[0002] Longan (Dimocarpus longan Lour.), also known as Guiyuan, is an evergreen large tree of the genus Dimocarpus in the family Sapindaceae. It is deeply loved by consumers because of its rich nutrition and sweet taste. In the fields of agricultural production and genetic breeding, the improvement and rapid propagation of longan varieties have always been research hotspots.

[0003] The traditional breeding method is to use explants for culture, such as cotyledons, young embryos, young stems, or seeds, etc. The regenerated plants obtained are mostly heterozygous diploids, which are difficult to meet the requirements of modern agriculture for high-efficiency and high-quality varieties. Therefore, using anther culture technology to induce longan anthers to produce haploid plants and then doubling them into homozygous diploids has become an important way to accelerate the breeding process and improve breeding efficiency.

[0004] Anther culture technology is one of the methods for culturing haploid plants. However, there are few reports on obtaining haploids through anther culture. Only Yang Yongqing et al. reported in 1984 that haploid plants were obtained using the anthers of longan "Dongbi" as materials, and there are no subsequent reports on related applications. Moreover, due to the high content of phenolic substances and small flower buds in longan plants, the current anther culture technology has developed slowly and has many deficiencies; for example, the anthers of different longan varieties have large differences in response to the culture medium, and there is a lack of a culture medium formula with strong universality; for another example, the browning degree of callus induction, proliferation, and differentiation during anther culture is high, resulting in low acquisition rates for each step. Summary of the Invention

[0005] In view of the above deficiencies in the prior art, the present invention aims to provide an efficient and stable longan anther culture medium by optimizing the culture medium formula, realizing the efficient induction, proliferation, and differentiation of longan anther callus, and then obtaining regenerated haploid plants; and with a low browning degree and a high induction rate. This technical solution aims to improve the longan breeding efficiency and accelerate the breeding process of excellent varieties.

[0006] To achieve the above object, the present invention can adopt the following technical solutions:

[0007] On the one hand, the present invention provides a callus induction medium for longan anther culture, comprising: 1 / 2MS medium, agar, sucrose, maltose, activated carbon, and 2,4-D, with concentrations of 2.47 g / L, 7 g / L, 20 g / L, 10 g / L, 0.3 g / L, and 0.2 - 0.8 mg / L respectively, and a pH value of 5.8.

[0008] On the other hand, the present invention provides a callus proliferation medium for longan anther culture, comprising: 1 / 2 MS medium, agar, sucrose, maltose, activated carbon, 2,4-D and 6-BA, with concentrations of 2.47 g / L, 7 g / L, 20 g / L, 10 g / L, 0.3 g / L, 0.2 - 0.5 mg / L and 0.05 - 0.1 mg / L respectively, and a pH value of 5.8.

[0009] On yet another aspect, the present invention provides a callus differentiation medium for longan anther culture, comprising: Medium 1, Medium 2, Medium 3 and Medium 4; wherein,

[0010] Medium 1 comprises: 1 / 2 MS medium, agar, sucrose, maltose, activated carbon, IBA and 6-BA, with concentrations of 2.47 g / L, 7 g / L, 20 g / L, 10 g / L, 0.3 g / L, 0.1 - 0.5 mg / L and 1.0 - 2.0 mg / L respectively, and a pH value of 5.8;

[0011] Medium 2 comprises: 1 / 2 MS medium, agar, sucrose, maltose, activated carbon, IBA and 6-BA, with concentrations of 2.47 g / L, 7 g / L, 20 g / L, 10 g / L, 0.3 g / L, 0.05 - 0.1 mg / L and 2.0 - 2.5 mg / L respectively, and a pH value of 5.8;

[0012] Medium 3 comprises: 1 / 2 MS medium, agar, sucrose, maltose and activated carbon, with concentrations of 2.47 g / L, 7 g / L, 20 g / L, 10 g / L and 0.3 g / L respectively, and a pH value of 5.8;

[0013] Medium 4 comprises: 1 / 2 MS medium, agar, sucrose, maltose, activated carbon, IAA and 6-BA, with concentrations of 2.47 g / L, 7 g / L, 20 g / L, 10 g / L, 0.3 g / L, 0.1 - 0.5 mg / L and 0.05 - 1 mg / L respectively, and a pH value of 5.8.

[0014] On yet another aspect, the present invention provides a method for cultivating longan haploid plants, comprising: (1) culturing longan anthers through the callus induction medium for longan anther culture in the present invention to obtain longan anther callus; (2) then proliferating the longan anther callus using the callus proliferation medium for longan anther culture in the present invention; (3) differentiating the proliferated callus using the callus differentiation medium for longan anther culture in the present invention to obtain embryoids or cluster buds; (4) culturing the embryoids or cluster buds to root to obtain longan haploid plants.

[0015] Preferably, in the above step (3), it includes: successively using Medium 1, Medium 2, Medium 3, and Medium 4 to differentiate the proliferated callus to obtain embryoids or cluster buds.

[0016] Preferably, in the above step (1), the culture conditions are a temperature of 24°C to 26°C and dark culture.

[0017] Preferably, in the above steps (2) to (4), the culture conditions include: a temperature of 24°C to 26°C, a light intensity of 1500 to 2000 Lux, and a light duration of 14 to 16 h.

[0018] It should be noted that the present invention includes 5 technical links: disinfection treatment of longan flower buds, explant stripping, anther callus induction, callus subculture proliferation, and callus differentiation; the method uses flower buds at the late uninucleate stage as materials, disinfects the flower buds with 75% alcohol for 10 s to 60 s, washes them several times with sterile water and then immerses them in a little sterile water, adsorbs harmful substances and inhibits the decomposition of phenolic substances by adding activated carbon to the medium, and subcultures once about every half month at the initial inoculation stage, etc., to ensure that the browning rate of the callus is within 20% and the induction rate is above 80%; and proliferates the callus, and a large amount of callus can be obtained in 2 to 3 months. Then, by adjusting the types and concentrations of auxin and cytokinin, embryoids or cluster buds with strong growth vigor are obtained, laying a foundation for further rooting culture.

[0019] It should also be noted that the present invention has been optimized in terms of anther selection period, anther pretreatment, sterilization method and duration, medium type, and growth regulator components, etc., to obtain a broad-spectrum anther culture callus induction, differentiation, and proliferation medium formula suitable for multiple longan varieties, and a large number of embryoids and cluster buds can be obtained in about 1 year, greatly shortening the time of anther culture.

[0020] The beneficial effects of the present invention at least include: the anther culture callus induction, proliferation, and differentiation medium provided by the present invention can achieve efficient induction, proliferation, and differentiation of longan anther callus, and then obtain regenerated haploid plants. Description of the Drawings

[0021] Figure 1 Shows the culture situation of the anther callus induction medium;

[0022] Figure 2 Shows the culture situations at different stages;

[0023] Figure 3 Shows the situation of inoculating anthers of different varieties at different inoculation times;

[0024] Figure 4 Shows the culture situations with different addition amounts of activated carbon as the adsorbent;

[0025] Figure 5 The culture conditions with PVP as the adsorbent at different addition amounts;

[0026] Figure 6 The culture conditions with citric acid as the adsorbent at different addition amounts;

[0027] Figure 7 The culture conditions with vitamin C as the adsorbent at different addition amounts. Specific embodiments

[0028] The examples given are for better illustration of the present invention, but the content of the present invention is not limited only to the examples given. Therefore, those skilled in the art who make non-essential improvements and adjustments to the implementation schemes based on the above-mentioned inventive content still fall within the protection scope of the present invention.

[0029] The terms used herein are only for describing specific embodiments and are not intended to limit the present disclosure. Unless having significantly different meanings in the context, the expressions in the singular form include the expressions in the plural form. As used herein, it should be understood that terms such as "including", "having", "containing" are intended to indicate the presence of features, numbers, operations, components, parts, elements, materials or combinations. The terms of the present invention are disclosed in the specification, and are not intended to exclude the possibility that one or more other features, numbers, operations, components, parts, elements, materials or their combinations may exist or can be added. As used herein, depending on the circumstances, " / " can be interpreted as "and" or "or".

[0030] To better understand the present invention, the content of the present invention will be further clarified below in conjunction with specific examples, but the content of the present invention is not limited only to the examples below.

[0031] I. Culture method

[0032] (I) Bud disinfection

[0033] Collect healthy and plump flower buds with a diameter of 2.20 mm to 2.55 mm at the uninucleate microspore stage as materials, bring them back to the laboratory and place them in a 4°C refrigerator for 48 h of cold shock; take out the materials before inoculation, rinse them several times with tap water, then disinfect them with a disinfectant (75% alcohol) for 30 s, and directly soak them in a little sterile water after washing 3 times with sterile water.

[0034] (II) Explant stripping

[0035] Carefully remove the sepals with a dissecting needle to expose the anthers, and then take out the anthers from the base of the anthers with a dissecting needle; try not to bring filaments during the operation process and avoid piercing the anther wall.

[0036] (III) Anther callus induction culture

[0037] The explants obtained above are cultured using anther callus induction medium, and the components of the medium are shown in Table 1 below; the culture conditions are: temperature 24°C - 26°C, no light.

[0038] Table 1 Components of Anther Callus Induction Medium

[0039] Component Content 1 / 2 MS medium 2.47 g / L Agar 7 g / L Sucrose 20 g / L Maltose 10 g / L Activated carbon 0.3 g / L 2,4-D (2,4-dichlorophenoxyacetic acid) 0.2 - 0.8 mg / L pH 5.8

[0040] After about 15 days, the anthers begin to swell, and then they are subcultured every 15 days. Callus grows after about 1 - 2 months.

[0041] At the same time, observe the browning situation. As Figure 1 shown, by calculating the browning rate, it is known that the browning rate is lower than 20%.

[0042] (IV) Proliferation Culture of Anther Callus

[0043] The callus obtained by culturing using anther callus induction medium above is cultured using anther callus proliferation medium, and the components of the medium are shown in Table 2 below; the culture conditions are: temperature 24°C - 26°C, light intensity 2000 Lux, light duration 16 h;

[0044] Table 2 Components of Anther Callus Proliferation Medium

[0045] Component Content 1 / 2 MS medium 2.47 g / L Agar 7 g / L Sucrose 20 g / L Maltose 10 g / L Activated carbon 0.3 g / L 2,4-D (2,4-dichlorophenoxyacetic acid) 0.2 - 0.5 mg / L 6-BA (6-benzylaminopurine) 0.05 - 0.1 mg / L pH 5.8

[0046] Subculture once every about 1 month, and a large amount of callus can be induced to proliferate after 2 - 3 months.

[0047] (V) Differentiation Culture of Anther Callus

[0048] The callus proliferated using anther callus proliferation medium is subcultured once every about 1 month using anther callus differentiation medium 1. After about 3 months of subculture, it is transferred to anther callus differentiation medium 2 and subcultured once every about 1 month until young embryoids or cluster buds grow, and then transferred to a hormone - free medium (anther callus differentiation medium 3) for 1 month; then transferred to anther callus differentiation medium 4 to promote the growth of embryoids or cluster buds, and subcultured once every about 1 month. After the embryoids or cluster buds grow strong, proceed to the next step of research.

[0049] The pH values of the above media (anther callus differentiation medium 1, anther callus differentiation medium 2, anther callus differentiation medium 3, and anther callus differentiation medium 4) are all 5.8; the culture conditions are all: temperature 24°C, light intensity 2000 Lux, light duration 16 h.

[0050] Among them, the compositions of anther callus differentiation medium 1, anther callus differentiation medium 2, anther callus differentiation medium 3, and anther callus differentiation medium 4 are shown in Table 3, Table 4, Table 5, and Table 6 respectively as follows.

[0051] Table 3 Composition of Anther Callus Differentiation Medium 1

[0052] Component Content 1 / 2 MS medium 2.47 g / L Agar 7 g / L Sucrose 20 g / L Maltose 10 g / L Activated carbon 0.3 g / L IBA (Indole-3-butyric acid) 0.1 - 0.5 mg / L 6-BA (6-benzylaminopurine) 1.0 - 2.0 mg / pH 5.8

[0053] Table 4 Composition of Anther Callus Differentiation Medium 2

[0054] Component Content 1 / 2 MS medium 2.47 g / L Agar 7 g / L Sucrose 20 g / L Maltose 10 g / L Activated carbon 0.3 g / L IBA (Indole-3-butyric acid) 0.05 - 0.1 mg / L 6-BA (6-benzylaminopurine) 2.0 - 2.5 mg / L pH 5.8

[0055] Table 5 Composition of Anther Callus Differentiation Medium 3

[0056] Component Content 1 / 2 MS medium 2.47 g / L Agar 7 g / L Sucrose 20 g / L Maltose 10 g / L Activated carbon 0.3 g / L pH 5.8

[0057] Table 6 Composition of Anther Callus Differentiation Medium 4

[0058] Component Content 1 / 2 MS medium 2.47 g / L Agar 7 g / L Sucrose 20 g / L Maltose 10 g / L Activated carbon 0.3 g / L IAA (Indole-3-acetic acid) 0.1 - 0.5 mg / L 6-BA (6-benzylaminopurine) 0.05 - 1 mg / L pH 5.8

[0059] The culture conditions at different stages are as Figure 2 shown. From left to right and top to bottom, the first row is anthers inducing callus; the first one in the second row is callus proliferation; the second and third ones in the second row and the first one in the third row are callus differentiation, differentiating into embryoids or cluster buds; the second one in the third row is rooting.

[0060] II. Conditions of Inoculating Different Anthers at Different Inoculation Times

[0061] According to the above culture method, different varieties of anthers were inoculated at different inoculation times. About 1 month after inoculation, the induction rate of anthers to induce callus (the induction rate of callus (%) = the number of explants producing callus / the total number of inoculated explants × 100%) was all above 80%. Specifically, as shown in Table 7 and Figure 3 shown, Figure 3 In A, only 1 out of 11 anthers did not induce callus, and the induction rate was 90.91%.

[0062] Table 7 Conditions of Inoculating Different Anthers at Different Inoculation Times

[0063]

[0064] III. Culture Conditions of Media Prepared with Different Adsorbents

[0065] In the following examples, the browning rate (%) = the number of explants with browning / the total number of inoculated explants × 100%.

[0066] (I) The adsorbent is activated carbon

[0067] In the above cultivation method, the adsorbent is set as activated carbon, and the other steps are the same as the above steps. The concentrations of activated carbon are set as 0.3 g / L, 0.5 g / L, and 1 g / L respectively. The cultivation conditions are as Figure 4 shown, and the browning rate results of activated carbon at different concentrations are shown in Table 8 below.

[0068] Table 8 Browning rate of the medium prepared with different concentrations of activated carbon after cultivation

[0069]

[0070]

[0071] From the above Table 8 and Figure 4 it can be known that the browning of the medium prepared with activated carbon as the adsorbent is relatively light, and the browning is the lightest (12.43%) at a concentration of 0.3 g / L, and the anthers are significantly swollen.

[0072] (2) The adsorbent is PVP

[0073] In the above cultivation method, the adsorbent is set as PVP, and the other steps are the same as the above steps. The concentrations of PVP are set as 0.51 g / L, 11 g / L, and 1.51 g / L respectively. The cultivation conditions are as Figure 5 shown, and the browning rate results of PVP at different concentrations are shown in Table 9 below.

[0074] Table 9 Browning rate of the medium prepared with different concentrations of PVP after cultivation

[0075] PVP concentration Browning rate 0.5 g / L 93.75％ 1 g / L 86.67％ 1.5 g / L 93.93％

[0076] From the above Table 9 and Figure 5 it can be known that the browning of the medium prepared with PVP as the adsorbent is serious, and the anthers are not significantly swollen.

[0077] (3) The adsorbent is citric acid

[0078] In the above cultivation method, the adsorbent is set as citric acid, and the other steps are the same as the above steps. The concentrations of citric acid are set as 1 g / L, 2.5 g / L, and 5 g / L respectively. The cultivation conditions are as Figure 6 shown, and the browning rate results of citric acid at different concentrations are shown in Table 10 below.

[0079] Table 10 Browning rate of the medium prepared with different concentrations of citric acid after cultivation

[0080] Citric acid concentration Browning rate 0.5 g / L 93.75％ 1 g / L 86.67％ 1.5 g / L 93.93％

[0081] From the above Table 10 and Figure 6It can be known that the browning of the culture medium prepared with citric acid as the adsorbent is serious, and the anther swelling is not obvious.

[0082] (4) The adsorbent is vitamin C (VC)

[0083] The adsorbent is set as VC, and the other steps are the same as the above steps. The concentrations of VC are set as 1 g / L, 2.5 g / L, and 5 g / L respectively. The culture conditions are as Figure 7 shown. The browning rate results of different concentrations of citric acid are shown in Table 11 below.

[0084] Table 11 Browning rate of the culture medium prepared with different VC concentrations

[0085] VC concentration Browning rate 0.1 g / L 26.69％ 0.5 g / L 20.69％ 0.8 g / L 27.62％

[0086] From the above Table 11 and Figure 7 it can be known that the browning of the culture medium prepared with VC as the adsorbent is light, but it cannot be sterilized by high temperature and high pressure. The method of filtration sterilization is adopted, and it is easy to carry bacteria during the operation process, resulting in an increase in the pollution rate.

[0087] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not restrictive. Although the present invention has been described in detail with reference to the preferred embodiments, those of ordinary skill in the art should understand that the technical solutions of the present invention can be modified or equivalently replaced without departing from the purpose and scope of the technical solutions of the present invention, and they should all be covered by the scope of the claims of the present invention.

Claims

1. A longan anther culture callus induction medium, characterized in that: include: The concentrations of 1 / 2MS medium, agar, sucrose, maltose, activated carbon and 2,4-D were 2.47 g / L, 7 g / L, 20 g / L, 10 g / L, 0.3 g / L and 0.2-0.8 mg / L, respectively, and the pH value was 5.

8.

2. A medium for callus proliferation in longan anther culture, characterized in that: include: The concentrations of 1 / 2MS medium, agar, sucrose, maltose, activated carbon, 2,4-D and 6-BA were 2.47 g / L, 7 g / L, 20 g / L, 10 g / L, 0.3 g / L, 0.2-0.5 mg / L and 0.05-0.1 mg / L, respectively, and the pH value was 5.

8.

3. A medium for callus differentiation of longan anther culture, characterized in that: include: Culture medium 1, culture medium 2, culture medium 3 and culture medium 4; wherein, Medium 1 includes: 1 / 2MS medium, agar, sucrose, maltose, activated carbon, IBA and 6-BA, and the concentrations thereof are 2.47 g / L, 7 g / L, 20 g / L, 10 g / L, 0.3 g / L, 0.1-0.5 mg / L and 1.0-2.0 mg / L, respectively, and the pH value thereof is 5.8; Medium 2 includes: 1 / 2MS medium, agar, sucrose, maltose, activated carbon, IBA and 6-BA, and the concentrations thereof are 2.47 g / L, 7 g / L, 20 g / L, 10 g / L, 0.3 g / L, 0.05-0.1 mg / L and 2.0-2.5 mg / L, respectively, and the pH value thereof is 5.8; Medium 3 includes: 1 / 2MS medium, agar, sucrose, maltose and activated carbon, with concentrations of 2.47 g / L, 7 g / L, 20 g / L, 10 g / L and 0.3 g / L, respectively, and a pH value of 5.8; Culture medium 4 includes: 1 / 2MS culture medium, agar, sucrose, maltose, activated carbon, IAA and 6-BA, with concentrations of 2.47 g / L, 7 g / L, 20 g / L, 10 g / L, 0.3 g / L, 0.1-0.5 mg / L and 0.05-1 mg / L, respectively, and its pH value is 5.

8.

4. A method for cultivating longan haploid plants, characterized in that: include: (1) culturing longan anthers using the longan anther culture callus induction medium according to claim 1 to obtain longan anther callus; (2) then proliferating the longan anther callus using the longan anther culture callus proliferation medium described in claim 2; (3) differentiating the proliferated callus using the longan anther culture callus differentiation medium described in claim 3 to obtain embryoids or cluster buds; (4) culturing the embryoids or bud clusters to root and obtain longan haploid plants; step (3) includes: using culture medium 1, culture medium 2, culture medium 3 and culture medium 4 in sequence to differentiate the proliferated callus tissue to obtain embryoids or bud clusters.

5. The method for cultivating longan haploid plants according to claim 4, characterized in that: In step (1), the culture conditions include: temperature of 24°C to 26°C, and dark culture.

6. The method for cultivating longan haploid plants according to claim 4 or 5, characterized in that: In steps (2) to (4), the culture conditions include: temperature 24°C to 26°C, light intensity 1500 to 2000 Lux, and light intensity 14 to 16 hours.

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