Hydrophytic tree callus induction culture medium, hydrophytic tree adventitious bud induction culture medium, hydrophytic tree adventitious root induction culture medium and application thereof

Abstract

The invention belongs to the technical field of plant culture, and particularly relates to a hydrocyan tree callus induction culture medium, a hydrocyan tree adventitious bud induction culture medium, a hydrocyan tree adventitious root induction culture medium and application thereof. The culture method based on the culture medium is not affected by seasons, the culture time is short, the propagation rate is high, the problems of low cutting survival rate, long propagation period, quality degradation and the like of the water-cyan trees at present are solved, and large-scale propagation of improved varieties of the water-cyan trees is achieved.

Description

Technical Field

[0001] The invention belongs to the technical field of plant cultivation, and in particular relates to a callus induction medium for water lilies, an adventitious bud induction medium for water lilies, an adventitious root induction medium for water lilies and applications thereof. Background Art

[0002] The endangered plant, the water lily, is a single genus of the genus Water lily in the family Kunlanchowiae. It has medicinal value, ecological value, ornamental value, and industrial value. In recent years, the population of Water lily in the wild habitat has gradually declined due to plant and human factors. Tissue culture technology can provide a large number of callus tissues and tissue culture seedlings, thereby helping the population of Water lily to recover, improve its germplasm resource bank, and lay the foundation for its genetic transformation system.

[0003] A tissue culture breeding method for water lily is currently disclosed, comprising the following steps: (1) selection and disinfection of explants (stem segments); (2) preparation of water lily extract; (3) primary induction culture to make the stem segments grow new buds; (4) secondary proliferation culture to make the new buds form clustered buds; (5) inoculating single buds of clustered buds into rooting medium to induce rooting; (6) domestication and transplantation of test tube seedlings. Although test tube seedlings are obtained by this method, the method has the following shortcomings: (1) the acquisition of explant stem segments is limited by time; (2) picking a large number of stem segments has an important impact on the subsequent growth of water lily plants; (3) the disinfection of stem segments uses toxic disinfectant mercuric chloride, which is harmful to the environment and experimenters. At the same time, there is no public report on the callus induction and differentiation system of water lily in China, so the callus induction system of water lily has not been established so far, which directly affects the development of water lily transgenic technology system based on this technology.

[0004] Therefore, there is a need to develop a technical method for callus induction and plant regeneration of water chestnut, so as to provide technical support for the rapid propagation and genetic transformation system of water chestnut. Summary of the invention

[0005] The present invention obtains sterile seedlings from water lily seeds, and then uses the cotyledons, radicles, hypocotyls, stem segments, etc. of the sterile seedlings as explants for experiments, thereby obtaining explants without time restrictions, and harvesting the seeds will not have a significant impact on the subsequent growth of water lily plants. Based on this, the present invention establishes a set of methods for water lily callus induction, adventitious bud induction, and adventitious root induction. The method can be used to efficiently obtain water lily callus, adventitious buds, and adventitious roots, solve the problem of water lily callus induction and plant regeneration, lay a technical foundation for water lily rapid propagation and transgenic technology, and promote the development of water lily tissue culture technology.

[0006] In order to achieve the above purpose, the following technical solutions can be adopted:

[0007] The present invention provides a callus induction culture medium for Pieris edulis, comprising a culture medium for woody plants and an additive, wherein the additive comprises 6-benzylaminopurine, 2,4-dichlorophenoxyacetic acid, sucrose and agar.

[0008] Preferably, the added amount of 6-benzylaminopurine is 0.4 mg / L-0.6 mg / L; and / or the added amount of 2,4-dichlorophenoxyacetic acid is 0.4 mg / L-0.6 mg / L; and / or the added amount of sucrose is 18 g / L-22 g / L; and / or the added amount of agar is 4 g / L-6 g / L.

[0009] Another aspect of the present invention provides a medium for inducing adventitious buds of Pieris edulis, comprising a medium for woody plants and additives, wherein the additives include 6-benzylaminopurine, naphthylacetic acid, sucrose and agar.

[0010] Preferably, the added amount of 6-benzylaminopurine is 0.5 mg / L-1.5 mg / L; and / or the added amount of naphthylacetic acid is 0.005 mg / L-0.015 mg / L; and / or the added amount of sucrose is 18 g / L-22 g / L; and / or the added amount of agar is 4 g / L-6 g / L.

[0011] In another aspect, the present invention provides a medium for inducing adventitious roots of water chestnuts, comprising a medium for woody plants and additives, wherein the additives include 6-benzylaminopurine, naphthylacetic acid, sucrose and agar.

[0012] Preferably, the added amount of 6-benzylaminopurine is 0.4 mg / L-0.6 mg / L; and / or the added amount of naphthylacetic acid is 0.09 mg / L-0.11 mg / L; and / or the added amount of sucrose is 18 g / L-22 g / L; and / or the added amount of agar is 4 g / L-6 g / L.

[0013] In another aspect, the present invention provides a method for inducing callus, adventitious buds and adventitious roots of Psoralea corylifolia, comprising:

[0014] (1) culturing the cotyledons of sterile seedlings of Ligusticum chuanxiong using the Ligusticum chuanxiong callus induction medium of the present invention to obtain Ligusticum chuanxiong callus;

[0015] (2) using the adventitious bud induction medium of the present invention to culture the stem segments with leaf axils of the Chinese hollyhock to obtain the adventitious buds of the Chinese hollyhock;

[0016] (3) The adventitious buds of the Chinese hollyhock are cultured using the Chinese hollyhock adventitious root induction medium of the present invention to obtain the adventitious roots of the Chinese hollyhock.

[0017] Preferably, the method satisfies one or more combinations of the following conditions:

[0018] (a) The culturing conditions of step (1) include culturing in the dark at 25°C ± 2°C;

[0019] (b) The culture conditions of step (2) include culturing at 25°C ± 2°C, a light intensity of 1500 lx, and 12 h / d;

[0020] (c) The culture conditions of step (3) include culturing at 25°C ± 2°C, a light intensity of 1500 lx, and 12 h / d.

[0021] Preferably, the cultivation of sterile seedling cotyledons of the water chestnut tree comprises: soaking and sterilizing water chestnut seeds, inoculating sterile seedling germination culture medium, and culturing under light to obtain sterile seedling cotyledons of the water chestnut tree.

[0022] Preferably, the sterile seedling germination medium comprises complete 1 / 4 MS medium, 7.5 g / L sucrose and 6.5 g / L agar.

[0023] The beneficial effects of the present invention include at least: the cultivation method based on the culture medium of the present invention is not affected by seasons, the cultivation time is short, and the reproduction rate is fast, which solves the current problems of low survival rate of water chestnut cuttings, long reproduction cycle, quality degradation, etc., and realizes large-scale breeding of water chestnut improved varieties. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 The effects of different types of explants on callus induction of Psoralea corylifolia were studied.

[0025] Figure 2 The conditions of callus induction from different types of explants; A, radicle; B, hypocotyl; C, cotyledon; D, January leaf; E, February leaf; F, March leaf;

[0026] Figure 3 The effects of different basic culture media on callus induction of Psoralea corylifolia.

[0027] Figure 4 The effects of different plant growth regulators on adventitious bud induction of the hypocotyl of the water hyacinth; 1-9 represent 9 treatment groups, as follows: 1: WPM+0.5mg / L6-BA+0.01mg / LNAA; 2:

[0028] WPM+0.5mg / L6-BA+0.05mg / LNAA; 3: WPM+0.5mg / L6-BA+0.1mg / LNAA; 4: WPM+1mg / L6-BA+0.01mg / LNAA; 5: WPM+1mg / L6-BA+0.05mg / LNAA; 6 : WPM+1mg / L6-BA+0.1mg / LNAA; 7: WPM+1.5mg / L6-BA+0.01mg / LNAA; 8: WPM+1.5mg / L6-BA+0.05mg / LNAA; 9: WPM+1.5mg / L6-BA+0.1mg / LNAA;

[0029] Figure 5 The process of adventitious bud induction in the hypocotyl of Psoralea corylifolia; A, 3 weeks; B, 4 weeks; C, 5 weeks; D, 6 weeks;

[0030] Figure 6 Different types of explants used to induce adventitious buds; from left to right, they are cotyledons, hypocotyls, and stem segments with leaves in the axils;

[0031] Figure 7 The effects of different types of explants on adventitious bud induction;

[0032] Figure 8 Induction of adventitious buds from different types of explants; A, cotyledon; B, hypocotyl; C, stem segment with leaf axils;

[0033] Fig. 9 The effects of different plant growth regulators on the adventitious roots induced by water chestnut; 1-9 represent 9 treatment groups, as follows: 1: WPM+0.5mg / L6-BA+0.01mg / LNAA; 2: WPM+0.5mg / L6-BA+0.05mg / LNAA; 3: WPM+0.5mg / L6-BA+0.1mg / LNAA; 4: WPM+1mg / L6-BA+0.01mg / LNAA; 5: WPM+1mg / L6-BA+0.05mg / LNAA; 6: WPM+1mg / L6-BA+0.1mg / LNAA; 7: WPM+1.5mg / L6-BA+0.01mg / LNAA; 8: WPM+1.5mg / L6-BA+0.05mg / LNAA; 9: WPM+1.5mg / L6-BA+0.1mg / LNAA;

[0034] Fig.10 For the complete plant obtained by cultivation. DETAILED DESCRIPTION

[0035] The examples are provided to better illustrate the present invention, but the present invention is not limited to the examples. Therefore, those skilled in the art may make non-essential improvements and adjustments to the implementation scheme according to the above invention content, which still fall within the protection scope of the present invention.

[0036] The terms used herein are only used to describe specific embodiments and are not intended to limit the present disclosure. Unless the context has a significantly different meaning, expressions in the singular include expressions in the plural. As used herein, it should be understood that terms such as "include", "have", "include" 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 combinations thereof may exist or may be added. As used herein, " / " may be interpreted as "and" or "or", depending on the circumstances.

[0037] In order to better understand the present invention, the content of the present invention is further explained below in conjunction with specific examples, but the content of the present invention is not limited to the following examples.

[0038] 1. Obtaining sterile seedlings

[0039] The water chestnut seeds were first treated with 75% alcohol for 30 seconds, rinsed with sterile water three times, and then sterilized with 8% NaClO for 15 minutes to obtain sterile seeds. The sterile seeds were inoculated in a complete 1 / 4 MS medium (purchased from Combis Biotech) providing nutrients (1 / 4 of all nutrients in the MS medium, and the agar content was unchanged at 6.5 g / L), and cultured in an environment of 25°C ± 2°C, a light intensity of 1500lx, and 12h / d to obtain sterile water chestnut seedlings.

[0040] Compared with the existing mercuric chloride disinfection, the seed disinfection method of the present invention has the advantages of non-toxic disinfectant, little damage to seeds and thorough disinfection; the seeds are disinfected with 8% NaClO for 15 minutes, which not only has no inhibitory effect on seed germination, but also has a low contamination rate of about 40%. In addition, the low trace elements, Fe salts and organic matter contained in the complete 1 / 4MS culture gene are more suitable for the germination and growth of water hyacinth seeds. At this time, the germination rate of the seeds is significantly higher than that of the MS culture medium, reaching about 90%.

[0041] 2. Callus Induction

[0042] Callus tissue was obtained by inoculating different types of explants (radicle, hypocotyl, cotyledon, January leaf, February leaf and March leaf) into callus induction medium (WPM + 0.5 mg / L 6-BA + 0.5 mg / L 2,4-D + sucrose 20 g / L + agar 5 g / L, pH = 5.8 ± 1) and inducing them in the dark for 1 month.

[0043] The results showed that the callus induction rate of cotyledons of P. truncatula as explants was about 90%, the callus induction rate of radicle and hypocotyl was about 50%, and the callus induction rate of January, February and March leaves was less than 40% ( Figure 1 The cotyledon not only has a high callus induction rate, but also has the best callus state, showing loose milky white callus with good differentiation potential (see Figure 2 ). Since the cotyledons of P. truncatum were young materials, their dedifferentiation ability was stronger, so the callus induction rate of cotyledons was significantly higher than that of radicles, hypocotyls, 1-month-old leaves, 2-month-old leaves and 3-month-old leaves.

[0044] In addition, the cotyledons of P. truncatula were inoculated into different callus induction media (WPM + 0.5 mg / L 6-BA + 0.5 mg / L 2,4-D + sucrose 20 g / L + agar 5 g / L, pH = 5.8 ± 1;

[0045] MS+0.5mg / L6-BA+0.5mg / L2,4-D+sucrose 20g / L+agar 5g / L, pH=5.8±1;

[0046] 1 / 2MS+0.5mg / L6-BA+0.5mg / L2,4-D+sucrose 20g / L+agar 5g / L, pH=5.8±1) was induced in the dark for 1 month to obtain callus tissue.

[0047] The results showed that the callus induction rates in WPM (purchased from Combi Biotech), MS (purchased from Combi Biotech) and 1 / 2MS medium (purchased from Combi Biotech) were 87.23%, 50.66% and 28.75%, respectively. It can be seen that the induction rate of cotyledon callus in MS and 1 / 2MS medium decreased significantly (see Figure 3 ), while WPM medium (a medium with low inorganic salt concentration, and nitrogen salt is mainly supplied in the form of calcium nitrate. Like seed germination, callus induction of Eupatorium truncatum is suitable for growth on low-salt medium) provides suitable nutrients for callus induction of Eupatorium truncatum cotyledons, and the callus induction rate is the highest.

[0048] 3. Adventitious Bud Induction

[0049] The sterile stem segments of the water lily seedlings are inoculated into an adventitious bud induction medium (WPM+1mg / L6-BA+0.01mg / LNAA+20g / L sucrose+5g / L agar, pH=5.8±1), and light induction is performed in an environment of 25°C±2°C, a light intensity of 1500lx, and 12h / d to obtain new buds. Compared with the existing water lily adventitious bud induction scheme (1 / 2MS+8g / L water lily extract+0.06g / L 6-BA+0.04g / L NAA+20g / L sucrose+4g / L nutrient solution), the present invention has the advantages of more streamlined formula (no need to extract water lily extract and nutrient solution), no pollution, and high adventitious bud induction rate.

[0050] In addition, adventitious bud induction medium containing different types and concentrations of plant growth regulators was used (treatment group 1: WPM + 0.5 mg / L6-BA + 0.01 mg / LNAA; treatment group 2: WPM + 0.5 mg / L6-BA + 0.05 mg / LNAA; treatment group 3: WPM + 0.5 mg / L6-BA + 0.1 mg / LNAA; treatment group 4: WPM + 1 mg / L6-BA + 0.01 mg / LNAA; treatment group 5: WPM + 1 mg / L6-BA + 0.05mg / LNAA; Treatment group 6: WPM+1mg / L6-BA+0.1mg / LNAA; Treatment group 7: WPM+1.5mg / L6-BA+0.01mg / LNAA; Treatment group 8: WPM+1.5mg / L6-BA+0.05mg / LNAA; Treatment group 9: WPM+1.5mg / L6-BA+0.1mg / LNAA) were used to induce adventitious buds on the hypocotyls of the water chestnut tree (cultured according to the above method), and the effects and growth conditions are shown in the figure. Figure 4 and Figure 5 The results showed that 6-BA and NAA had a significant effect on the induction of adventitious buds from the hypocotyls of sterile seedlings of Psoralea corylifolia. Treatment groups 1 and 4 were most suitable for adventitious bud induction, with an induction rate of about 15%. Treatment groups 3, 5, 6, 8, and 9 were unable to induce adventitious buds ( Figure 4 ); In the treatment group where adventitious buds were successfully induced, yellow-green callus was induced from the hypocotyl of the water hyacinth at the 3rd week, callus differentiated into buds at the 4th week, buds differentiated into adventitious buds at the 5th week, and adventitious buds further grew in the 6th week ( Figure 5 ).

[0051] Next, different types of explants (cotyledons, hypocotyls, and axillary stem segments with leaves ( Figure 6)) were inoculated in adventitious bud induction medium (WPM+1mg / L6-BA+0.01mg / LNAA+20g / L sucrose+5g / L agar, pH=5.8±1) (the highest induction rate was in treatment group 1, followed by treatment group 4, and there was no significant difference between the two treatment groups, but a high ratio of NAA to 6BA was more conducive to in vitro adventitious bud differentiation, so this formula was selected for the experiment), and light induction was carried out at 25℃±2℃, light intensity of 1500lx, 12h / d to obtain adventitious buds; the results showed that the adventitious bud induction rate of the stem segment with leaf axils of the aseptic seedlings of the water lily was 100%, the adventitious bud induction rate of the hypocotyl was 16.66%, and the adventitious bud induction rate of the cotyledon was 0% ( Figure 7 ), and the new shoots induced from the stem segments with leaf axils are very strong (see Figure 8 ), is an excellent material for subsequent induction of adventitious roots to form complete plants. Because the endogenous hormones in the axils of stem segments are similar to those produced by new buds, the induction of new buds is quickly initiated after a certain concentration of auxin and cytokinin are added exogenously. In addition, the WPM medium with a low concentration of inorganic salts also provides a suitable induction environment for the induction of adventitious buds in the stem segments of the water lily.

[0052] 4. Adventitious root induction

[0053] The method is to induce new shoots of about 2 cm in height from the stem segments of sterile water lily seedlings and inoculate them into adventitious root induction medium (WPM+0.5mg / L6-BA+0.1mg / LNAA+20g / L sucrose+5g / L agar, pH=5.8±1), and perform light induction at 25°C±2°C, 1500lx light intensity, 12h / d to obtain adventitious roots. Compared with the existing water lily adventitious bud induction scheme (1 / 4MS+0.5g / L water lily extract+0.03g / L IBA+0.02g / L NAA+15g / L sucrose+3g / L agar), this scheme has the advantages of more concise formula (no need to extract water lily extract) and high adventitious root induction rate.

[0054] In addition, adventitious root induction medium containing different types and concentrations of plant growth regulators was used (treatment group 1: WPM + 0.5 mg / L6-BA + 0.01 mg / LNAA; treatment group 2: WPM + 0.5 mg / L6-BA + 0.05 mg / LNAA; treatment group 3: WPM + 0.5 mg / L6-BA + 0.1 mg / LNAA; treatment group 4: WPM + 1 mg / L6-BA + 0.01 mg / LNAA; treatment group 5: WPM + 1 mg / L6-BA + 0.05 mg / LNAA; treatment group 6: WPM + 1 mg / L6-BA + 0.1 mg / LNAA). AA; treatment group 7: WPM+1.5mg / L6-BA+0.01mg / LNAA; treatment group 8: WPM+1.5mg / L6-BA+0.05mg / LNAA; treatment group 9: WPM+1.5mg / L6-BA+0.1mg / LNAA) were light-induced to obtain adventitious roots (the method was carried out according to the above method); the results showed that the adventitious root induction rate (differentiation rate) of the water plantain in treatment group 3 was close to 100%, the induction rates of treatment groups 2 and 6 were about 90%, the induction rates of treatment groups 1, 5, 8 and 9 were about 75%, the induction rate of treatment group 4 was about 40%, and the induction rate of treatment group 7 was the lowest, less than 20% ( Fig. 9 ), and the complete plants obtained from the induced treatment group 3 were very strong ( Fig.10 ), which laid a good foundation for the subsequent acclimatization and transplantation of the whole plant. Consistent with the above process, the WPM medium with low inorganic salt concentration also provided a suitable induction environment for the adventitious root induction of adventitious buds of the water hyacinth.

[0055] Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present invention rather than to limit it. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solution of the present invention can be modified or replaced by equivalents without departing from the purpose and scope of the technical solution of the present invention, which should be covered by the scope of the claims of the present invention.

Claims

1. A medium for inducing callus tissue of Pieris edulis, characterized in that: The invention comprises a culture medium and additives for woody plants, wherein the additives comprise 6-benzylaminopurine, 2,4-dichlorophenoxyacetic acid, sucrose and agar.

2. The callus induction medium of claim 1, characterized in that: The added amount of 6-benzylaminopurine is 0.4 mg / L-0.6 mg / L; and / or the added amount of 2,4-dichlorophenoxyacetic acid is 0.4 mg / L-0.6 mg / L; and / or the added amount of sucrose is 18 g / L-22 g / L; and / or the added amount of agar is 4 g / L-6 g / L.

3. A medium for inducing adventitious buds of Pieris edulis, characterized in that: The invention comprises a culture medium and additives for woody plants, wherein the additives comprise 6-benzylaminopurine, naphthylacetic acid, sucrose and agar.

4. The adventitious bud induction medium of Pieris edulis according to claim 3, characterized in that: The added amount of 6-benzylaminopurine is 0.5mg / L-1.5mg / L; and / or the added amount of naphthylacetic acid is 0.005mg / L-0.015mg / L; and / or the added amount of sucrose is 18g / L-22g / L; and / or the added amount of agar is 4g / L-6g / L.

5. A medium for inducing adventitious roots of Pieris edulis, characterized in that: The invention comprises a culture medium and additives for woody plants, wherein the additives comprise 6-benzylaminopurine, naphthylacetic acid, sucrose and agar.

6. The adventitious root induction medium of Pieris edulis according to claim 5, characterized in that The added amount of 6-benzylaminopurine is 0.4mg / L-0.6mg / L; and / or the added amount of naphthylacetic acid is 0.09mg / L-0.11mg / L; and / or the added amount of sucrose is 18g / L-22g / L; and / or the added amount of agar is 4g / L-6g / L.

7. A method for inducing callus, adventitious buds and adventitious roots of Pieris edulis, characterized in that: include: (1) culturing the cotyledons of sterile seedlings of Liriodendron edulis using the Liriodendron edulis callus induction medium described in any one of claims 1 to 2 to obtain Liriodendron edulis callus; (2) using the adventitious bud induction medium of any one of claims 3 to 4 to culture the leaf axillary stem segments of the Chinese hollyhock tree to obtain adventitious buds of the Chinese hollyhock tree; (3) The adventitious buds of the Chinese hollyhock are cultured using the Chinese hollyhock adventitious root induction medium according to any one of claims 5 to 6 to obtain the Chinese hollyhock adventitious roots.

8. The method according to claim 7, characterized in that The method meets one or more combinations of the following conditions: (a) The culturing conditions of step (1) include culturing in the dark at 25°C ± 2°C; (b) The culture conditions of step (2) include culturing at 25°C ± 2°C, a light intensity of 1500 lx, and 12 h / d; (c) The culture conditions of step (3) include culturing at 25°C ± 2°C, a light intensity of 1500 lx, and 12 h / d.

9. The method according to claim 7 or 8, characterized in that: The cultivation of the cotyledons of the aseptic seedlings of the water lily comprises the following steps: soaking the water lily seeds, sterilizing them, inoculating the aseptic seedling germination medium, and culturing them under light to obtain the cotyledons of the aseptic seedlings of the water lily.

10. The method according to claim 9, characterized in that The sterile seedling germination medium includes complete 1 / 4 MS medium, 7.5 g / L sucrose and 6.5 g / L agar.

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