An efficient regeneration method using the leaf nodes of Gmelina arborea as explants

Abstract

The present invention provides an efficient regeneration method using the cotyledon nodes of Anthocephalus chinensis as explants, which relates to the technical field of plant tissue culture and comprises the following steps: S1: Culturing aseptic seedlings of superior Anthocephalus chinensis trees, and selecting the cotyledon nodes of the seedlings; S2: Placing the cotyledon nodes in a callus induction medium for culture to obtain callus; S3: Placing the callus in an adventitious bud induction medium for culture to obtain effective buds; S4: Placing the effective buds in a rooting medium for culture to obtain Anthocephalus chinensis seedlings. Among them, the callus induction medium comprises: MS, 3-7 mg / L TDZ, 0.1-0.2 mg / L NAA, 25-30 g / L sucrose and 5-7 g / L agar. In the present invention, the callus induction rate of the cotyledon nodes of Anthocephalus chinensis is as high as 100%, and the budding effect is good. By using the callus of the cotyledon nodes of Anthocephalus chinensis for in vitro regeneration, it can be used for the preservation of Anthocephalus chinensis germplasm resources and large-scale seedling production, and also lays a foundation for transgenic research.

Description

Technical Field

[0001] The present invention relates to the technical field of plant tissue culture, and in particular to an efficient regeneration method using the cotyledon node of Anthocephalus chinensis as an explant. Background Art

[0002] At present, the establishment of the regeneration system of Anthocephalus chinensis is mainly achieved through cotyledons, hypocotyls and young leaves. Huang Hao et al. used cotyledons and hypocotyls as explants and cultured them on DCR medium supplemented with 2.5 mg / L TDZ + 0.05 mg / L NAA to induce high-quality callus; then they were transferred to MS medium supplemented with 5.0 mg / L 6-BA and 0.05 mg / L NAA for bud induction, and the number of effective buds per explant reached 35.2, but the time-consuming was long, taking 10 weeks. Li Jingjian et al. used young leaves as explants and also induced good callus on MS medium supplemented with 3.0 mg / L TDZ + 0.1 mg / L 2,4-D + 0.05 mg / L NAA, and then transferred them to MS medium containing 0.5 mg / L 6-BA + 0.05 mg / L NAA, and the number of effective buds per explant reached 5.9, but the number of effective buds was small.

[0003] At present, there is no report on the method of inducing with the cotyledon node of Anthocephalus chinensis as an explant. There are dormant axillary buds in the cotyledon node, and axillary bud germination is likely to occur during callus induction, thus affecting the development of callus. Therefore, it is necessary to inhibit the germination of dormant axillary buds and improve the callus induction rate of the cotyledon node. Summary of the Invention

[0004] The purpose of the present invention is to provide an efficient regeneration method using the cotyledon node of Anthocephalus chinensis as an explant. In the present invention, the callus induction rate of the cotyledon node of Anthocephalus chinensis is as high as 100%, and the budding effect is good. Through in vitro regeneration mediated by the callus of the cotyledon node of Anthocephalus chinensis, it can be used for the preservation of germplasm resources of Anthocephalus chinensis and large-scale production of seedlings, and at the same time, it also lays a foundation for transgenic research.

[0005] In order to achieve the above purpose, the present invention adopts the following technical solutions:

[0006] The present invention provides an efficient regeneration method using the cotyledon node of Anthocephalus chinensis as an explant, comprising the following steps:

[0007] S1: Culturing aseptic Anthocephalus chinensis elite tree seedlings and selecting the cotyledon nodes of the seedlings;

[0008] S2: Placing the cotyledon nodes in a callus induction medium for culture to obtain callus;

[0009] S3: Placing the callus in an adventitious bud induction medium for culture to obtain effective buds;

[0010] S4: Place the effective buds in a rooting medium for cultivation to obtain Anthocephalus chinensis seedlings;

[0011] Among them, the callus induction medium includes: MS, 3 - 7 mg / L TDZ, 0.1 - 0.2 mg / L NAA, 25 - 30 g / L sucrose, and 5 - 7 g / L agar.

[0012] Further, based on the above technical solution, the callus induction medium includes: MS, 5 mg / L TDZ, 0.15 mg / L NAA, 25 - 30 g / L sucrose, and 5 - 7 g / L agar.

[0013] Further, based on the above technical solution, the adventitious bud induction medium includes: MS, 3 - 7 mg / L 6 - BA, 0.03 - 0.07 mg / L NAA, 25 - 30 g / L sucrose, and 5 - 7 g / L agar;

[0014] Preferably, the adventitious bud induction medium includes: MS, 5 mg / L 6 - BA, 0.05 mg / L NAA, 25 - 30 g / L sucrose, and 5 - 7 g / L agar.

[0015] Further, based on the above technical solution, the rooting medium includes: 1 / 2MS, 0.05 - 0.15 mg / L IBA, 0.01 - 0.08 mg / L NAA, 25 - 30 g / L sucrose, and 5 - 7 g / L agar;

[0016] Preferably, the rooting medium includes: 1 / 2MS, 0.1 mg / L IBA, 0.05 mg / L NAA, 25 - 30 g / L sucrose, and 5 - 7 g / L agar.

[0017] Further, based on the above technical solution, the MS includes 1900 mg / L potassium nitrate, 332.2 mg / L calcium chloride, 1650 mg / L ammonium nitrate, 180.7 mg / L magnesium sulfate, 170 mg / L potassium dihydrogen phosphate, 0.014 mg / L cobalt chloride, 0.016 mg / L copper sulfate, 6.2 mg / L boric acid, 15.1 mg / L manganese sulfate, 0.21 mg / L sodium molybdate, 4.8 mg / L zinc sulfate, 0.83 mg / L potassium iodide, 27.8 mg / L FeSO4·7H2O, 37.25 mg / L Na2 - EDTA, 2.0 mg / L glycine, 100 mg / L inositol, 0.1 mg / L thiamine VB1, 0.50 mg / L nicotinic acid, 0.5 mg / L pyridoxine VB6.

[0018] Further, based on the above technical solution, in step S1, the cultivation of sterile Anthocephalus chinensis elite tree seedlings includes the following steps:

[0019] S11: Soak Anthocephalus chinensis seeds in water for 12 - 24 h;

[0020] S12: Transfer the seeds soaked in water to 75% - 80% alcohol and soak for 40 - 70 s;

[0021] S13: Wash the seeds soaked in alcohol with sterile water and sterilize them with 25% - 30% sodium hypochlorite for 3 - 7 min to obtain sterilized seeds;

[0022] S14: After washing the sterilized seeds with sterile water, transfer the sterilized seeds to MS basal medium for cultivation to obtain sterile Anthocephalus chinensis elite tree seedlings.

[0023] Further, on the basis of the above technical solution, in step S14, the cultivation conditions in the MS basal medium are:

[0024] Light intensity is 1800 - 2300 lx, light time is 10 - 14 h / d, and temperature is 23 - 27 °C.

[0025] Further, on the basis of the above technical solution, in step S2, the cultivation conditions of the callus induction medium are: light intensity is 1800 - 2300 lx, light time is 10 - 14 h / d, temperature is 23 - 27 °C, pH is 5.8 - 6.0, and time is 4 weeks;

[0026] And / or, the cotyledon node is the cotyledon node with petiole, and the length is 3 - 7 mm.

[0027] Further, on the basis of the above technical solution, in step S3, the cultivation conditions of the adventitious bud induction medium are: light intensity is 1800 - 2300 lx, light time is 10 - 14 h / d, temperature is 23 - 27 °C, pH is 5.8 - 6.0, and time is 4 weeks;

[0028] And / or, the length of the effective bud is greater than 1 cm.

[0029] Further, on the basis of the above technical solution, in step S4, the cultivation conditions of the rooting medium are: light intensity is 1800 - 2300 lx, light time is 10 - 14 h / d, temperature is 23 - 27 °C, pH is 5.8 - 6.0, and time is 20 days.

[0030] An efficient regeneration method using Anthocephalus chinensis cotyledon node as explant provided by the present invention has the following beneficial effects:

[0031] 1. The present invention provides an efficient regeneration method using the cotyledon nodes of Gmelina arborea as explants. This method is simple, efficient, and reproducible for callus-mediated in vitro regeneration of cotyledon nodes. According to the study on the influencing factors of plant growth regulators on callus formation, the results show that TDZ is the best cytokinin for inducing callus from cotyledon node explants. In the MS medium, the MS medium supplemented with 5 mg / L TDZ + 0.15 mg / L NAA has the best callus induction effect, with a callus induction rate of 100%, and the germination rate of dormant axillary buds is 0%. The induced callus is transferred to a medium containing 5 mg / L 6-BA + 0.05 mg / L NAA for adventitious bud induction, and effective buds can be obtained in only 8 weeks, with an average number of effective buds per explant reaching 36.

[0032] 2. The present invention has developed a simple, efficient, and reproducible protocol. In this method, the callus induction rate of the cotyledon nodes of Gmelina arborea is as high as 100%, and the callus has good physiological state and excellent budding effect. By using the callus of the cotyledon nodes of Gmelina arborea for in vitro regeneration, it can be used for the conservation of Gmelina arborea germplasm resources and large-scale seedling production, and also lays a foundation for transgenic research.

[0033] 3. The regeneration system of the cotyledon nodes of Gmelina arborea provided by the present invention has a higher budding efficiency and shorter overall time required compared to other explants of Gmelina arborea. It can not only be used for the rapid propagation of Gmelina arborea seedlings and the conservation of high-quality resources, but also lays a foundation for genetic transformation research. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] In order to more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the following will briefly introduce the drawings required for the description of the specific embodiments or the prior art. Obviously, the following drawings are some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can also be obtained based on these drawings.

[0035] Figure 1 Schematic diagram of cutting the cotyledon nodes of sterile Gmelina arborea elite tree seedlings as explants for induction test provided in Example 1 of the present invention;

[0036] Figure 2 Photo of the callus obtained after culturing in the callus induction medium for 4 weeks provided in Example 1 of the present invention;

[0037] Figure 3 Photo of the effective buds obtained after culturing in the adventitious bud induction medium for 4 weeks provided in Example 1 of the present invention;

[0038] Figure 4 Photo of a single effective bud obtained after culturing in the adventitious bud induction medium for 4 weeks provided in Example 1 of the present invention;

[0039] Figure 5 This is a photo of the effective bud rooting obtained after culturing in the rooting medium for 20 days provided in Example 1 of the present invention;

[0040] Figure 6 This is a photo of the Anthocephalus chinensis seedlings obtained after culturing in the rooting medium for 20 days provided in Example 1 of the present invention. Detailed implementation manners

[0041] To make the objectives, technical solutions and advantages of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Those skilled in the art should understand that the described embodiments are only for helping to understand the present invention and should not be regarded as specific limitations on the present invention. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts fall within the scope of protection of the present invention. The process parameters without specific conditions noted in the following embodiments are usually in accordance with conventional conditions.

[0042] In the ranges disclosed in the present invention, the endpoints and any values are not limited to the exact ranges or values. These ranges or values should be understood to include values close to these ranges or values. For numerical ranges, between the endpoint values of each range, between the endpoint values and individual point values of each range, and between individual point values, they can be combined with each other to obtain one or more new numerical ranges, and these numerical ranges should be regarded as specifically disclosed in the present invention.

[0043] According to the first aspect of the present invention, there is provided an efficient regeneration method using the cotyledon node of Anthocephalus chinensis as an explant, including the following steps:

[0044] S1: Cultivate sterile Anthocephalus chinensis elite tree seedlings and select the cotyledon nodes of the seedlings;

[0045] S2: Place the cotyledon nodes in a callus induction medium for cultivation to obtain callus;

[0046] S3: Place the callus in an adventitious bud induction medium for cultivation to obtain effective buds;

[0047] S4: Place the effective buds in a rooting medium for cultivation to obtain Anthocephalus chinensis seedlings.

[0048] The present invention develops a regeneration system for the cotyledon nodes of Anthocephalus chinensis, which has a higher bud emergence efficiency compared to other explants of Anthocephalus chinensis, requires a shorter overall time, can better induce callus, inhibit axillary bud germination, has a callus induction rate of 100%, and the physiological state of the callus is good.

[0049] As an alternative embodiment of the present invention, the callus induction medium comprises: MS, 3 - 7 mg / L (such as 4 mg / L, 5 mg / L, 6 mg / L, etc.) TDZ, 0.1 - 0.2 mg / L (such as 0.12 mg / L, 0.13 mg / L, 0.14 mg / L, 0.15 mg / L, 0.16 mg / L, 0.17 mg / L, 0.18 mg / L, 0.19 mg / L, etc.) NAA, 25 - 30 g / L (such as 26 g / L, 27 g / L, 28 g / L, 29 g / L, etc.) sucrose and 5 - 7 g / L (such as 5.5 g / L, 6 g / L, 6.5 g / L, etc.) agar;

[0050] Preferably, the callus induction medium comprises: MS, 5 mg / L TDZ, 0.15 mg / L NAA, 25 - 30 g / L sucrose and 5 - 7 g / L agar.

[0051] Specifically, TDZ is a phenylurea - type cytokinin. Its biological effects have been considered superior to or similar to the most active adenine - type cytokinins. It has been proven to be the most critical factor in inducing somatic embryogenesis and bud regeneration.

[0052] In the present invention, TDZ is added to the callus induction medium because TDZ can effectively induce callus formation from the cotyledons and hypocotyls of Neolamarckia cadamba, and enable adventitious buds to be produced from the callus.

[0053] However, if the concentration of TDZ is too low, the explants will not be able to form callus; if the concentration of TDZ is too high, it will have a toxic inhibitory effect on the cotyledon nodes of Neolamarckia cadamba. Therefore, the concentration of TDZ needs to be strictly controlled.

[0054] Specifically, NAA is further added to the callus induction medium in the present invention because when the concentration of TDZ is too low, the combination of NAA will increase the callus induction rate of the cotyledon nodes of Neolamarckia cadamba, and even enable the explants to directly germinate and root. However, the concentration of NAA will also affect the callus induction rate of the cotyledon nodes of Neolamarckia cadamba. If the added concentration of NAA is too high, it will strengthen the toxic inhibitory effect of TDZ on the cotyledon nodes of Neolamarckia cadamba; if the added concentration of NAA is too low, the effect on TDZ will not be significant, thus affecting the formation of callus, and instead increasing the axillary bud germination and even rooting.

[0055] Furthermore, the MS medium is selected in the present invention because the MS medium has high contents of nitrates, potassium and ammonium, which helps to accelerate the growth of callus.

[0056] In summary, by strictly defining TDZ at a concentration of 5 mg / L + NAA at a concentration of 0.15 mg / L + MS in the present invention, a synergistic effect is produced among MS, TDZ and NAA, so that the callus induction rate of the cotyledon node explants of Neolamarckia cadamba by the present invention reaches 100%.

[0057] As an alternative embodiment of the present invention, the adventitious bud induction medium comprises: MS, 3 - 7 mg / L (such as 4 mg / L, 5 mg / L, 6 mg / L, etc.) 6 - BA, 0.03 - 0.07 mg / L (such as 0.04 mg / L, 0.05 mg / L, 0.06 mg / L, etc.) NAA, 25 - 30 g / L (such as 26 g / L, 27 g / L, 28 g / L, 29 g / L, etc.) sucrose and 5 - 7 g / L (such as 5.5 g / L, 6 g / L, 6.5 g / L, etc.) agar;

[0058] Preferably, the adventitious bud induction medium comprises: MS, 5 mg / L 6 - BA, 0.05 mg / L NAA, 25 - 30 g / L sucrose and 5 - 7 g / L agar.

[0059] Specifically, the adventitious buds obtained after callus induction of the cotyledon node explants of Strychnos nux - vomica L. have the largest number of effective buds in the adventitious bud induction medium containing MS, 6 - BA at a concentration of 3 - 7 mg / L, and NAA at a concentration of 0.03 - 0.07 mg / L.

[0060] As an alternative embodiment of the present invention, the rooting medium comprises: 1 / 2 MS, 0.05 - 0.15 mg / L (such as 0.06 mg / L, 0.07 mg / L, 0.08 mg / L, 0.09 mg / L, 0.1 mg / L, 0.12 mg / L, 0.13 mg / L, 0.14 mg / L, etc.) IBA, 0.01 - 0.08 mg / L (such as 0.02 mg / L, 0.03 mg / L, 0.04 mg / L, 0.04 mg / L, 0.06 mg / L, 0.07 mg / L, etc.) NAA, 25 - 30 g / L (such as 26 g / L, 27 g / L, 28 g / L, 29 g / L, etc.) sucrose and 5 - 7 g / L (such as 5.5 g / L, 6 g / L, 6.5 g / L, etc.) agar;

[0061] Preferably, the rooting medium comprises: 1 / 2 MS, 0.1 mg / L IBA, 0.05 mg / L NAA, 25 - 30 g / L sucrose and 5 - 7 g / L agar.

[0062] Specifically, the adventitious buds obtained after callus induction of the cotyledon node explants of Strychnos nux - vomica L. have the highest rooting rate and the largest number of roots in the adventitious root induction medium containing 1 / 2 MS, IBA at a concentration of 0.05 - 0.15 mg / L, and NAA at a concentration of 0.01 - 0.08 mg / L.

[0063] As an alternative embodiment of the present invention, the MS comprises 1900 mg / L potassium nitrate, 332.2 mg / L calcium chloride, 1650 mg / L ammonium nitrate, 180.7 mg / L magnesium sulfate, 170 mg / L potassium dihydrogen phosphate, 0.014 mg / L cobalt chloride, 0.016 mg / L copper sulfate, 6.2 mg / L boric acid, 15.1 mg / L manganese sulfate, 0.21 mg / L sodium molybdate, 4.8 mg / L zinc sulfate, 0.83 mg / L potassium iodide, 27.8 mg / L FeSO4·7H2O, 37.25 mg / L Na2-EDTA, 2.0 mg / L glycine, 100 mg / L inositol, 0.1 mg / L thiamine VB1, 0.50 mg / L nicotinic acid, 0.5 mg / L pyridoxine VB6.

[0064] As an alternative embodiment of the present invention, in step S1, culturing the aseptic superior tree seeds of Anthocephalus chinensis comprises the following steps:

[0065] S11: Soak the Anthocephalus chinensis seeds in water for 12 - 24 h (such as 14 h, 16 h, 18 h, 20 h, 22 h, etc.);

[0066] S12: Transfer the seeds soaked in water to alcohol with a concentration of 75% - 80% (such as 76%, 77%, 78%, 79%, etc.) and soak for 40 - 70 s (such as 50 s, 60 s, etc.);

[0067] S13: Wash the seeds soaked in alcohol with sterile water and sterilize them with sodium hypochlorite with a concentration of 25% - 30% (such as 26%, 27%, 28%, 29%, etc.) for 3 - 7 min (4 min, 5 min, 6 min, etc.) to obtain sterilized seeds;

[0068] S14: After washing the sterilized seeds with sterile water, transfer the sterilized seeds to the MS basal medium for culturing to obtain aseptic superior tree seeds of Anthocephalus chinensis.

[0069] As an alternative embodiment of the present invention, in step S14, the culture conditions in the MS basal medium are:

[0070] The light intensity is 1800 - 2300 lx (such as 1900 lx, 2000 lx, 2100 lx, 2200 lx, etc.), the light time is 10 - 14 h / d (such as 11 h / d, 12 h / d, 13 h / d, etc.), and the temperature is 23 - 27 °C (such as 24 °C, 25 °C, 26 °C, etc.).

[0071] As an alternative embodiment of the present invention, in step S2, the culture conditions of the callus induction medium are as follows: the light intensity is 1800 - 2300 lx (such as 1900 lx, 2000 lx, 2100 lx, 2200 lx, etc.), the light time is 10 - 14 h / d (such as 11 h / d, 12 h / d, 13 h / d, etc.), the temperature is 23 - 27 °C (such as 24 °C, 25 °C, 26 °C, etc.), the pH is 5.8 - 6.0, and the time is 4 weeks;

[0072] And / or, the cotyledon node is the cotyledon node with petiole, and the length is 3 - 7 mm.

[0073] Specifically, the present invention selects the cotyledon node with petiole for callus induction because the callus obtained from the cotyledon node with petiole of Neolamarckia cadamba has the best effect in inducing adventitious buds after being cultured in the adventitious bud induction medium, and effective adventitious buds can be obtained in only 4 weeks, and the average number of effective buds per explant is about 36.

[0074] As an alternative embodiment of the present invention, in step S3, the culture conditions of the adventitious bud induction medium are as follows: the light intensity is 1800 - 2300 lx (such as 1900 lx, 2000 lx, 2100 lx, 2200 lx, etc.), the light time is 10 - 14 h / d (such as 11 h / d, 12 h / d, 13 h / d, etc.), the temperature is 23 - 27 °C (such as 24 °C, 25 °C, 26 °C, etc.), the pH is 5.8 - 6.0, and the time is 4 weeks;

[0075] And / or, the length of the effective bud is greater than 1 cm.

[0076] As an alternative embodiment of the present invention, in step S4, the culture conditions of the rooting medium are as follows: the light intensity is 1800 - 2300 lx (such as 1900 lx, 2000 lx, 2100 lx, 2200 lx, etc.), the light time is 10 - 14 h / d (such as 11 h / d, 12 h / d, 13 h / d, etc.), the temperature is 23 - 27 °C (such as 24 °C, 25 °C, 26 °C, etc.), the pH is 5.8 - 6.0, and the time is 20 days.

[0077] As an alternative embodiment of the present invention, before use, the MS basal medium, the callus induction medium, the adventitious bud induction medium, and the rooting medium need to be autoclaved at 120 - 125 °C (such as 121 °C, 122 °C, 123 °C, 124 °C, etc.) and 0.08 - 0.12 MPa (such as 0.09 MPa, 0.1 MPa, 0.11 MPa, etc.) for 15 - 25 min (such as 16 min, 18 min, 20 min, 22 min, 24 min, etc.).

[0078] The present invention will be further described in detail below in conjunction with specific examples and comparative examples.

[0079] It should be noted that the Anthocephalus chinensis plus tree seeds used in the examples and comparative examples provided by the present invention were collected from perennial Anthocephalus chinensis on the teaching and research base of South China Agricultural University in Zengcheng, Guangzhou.

[0080] Example 1

[0081] S1: Cultivate aseptic Anthocephalus chinensis plus tree seedlings and select the cotyledon nodes of the seedlings:

[0082] S11: Soak the Anthocephalus chinensis seeds in water for 24 h in a laminar flow hood;

[0083] S12: Transfer the seeds soaked in water to 75% alcohol and soak for 60 s;

[0084] S13: Wash the seeds soaked in alcohol 3 - 5 times with sterile water and sterilize with 25% sodium hypochlorite for 5 min to obtain sterilized seeds;

[0085] S14: After washing the sterilized seeds 3 - 5 times with sterile water, transfer the sterilized seeds to the MS basal medium obtained by autoclaving at 121°C and 0.1 MPa for 20 min for cultivation to obtain aseptic Anthocephalus chinensis plus tree seedlings. After the seedlings grow 1 - 2 pairs of true leaves, cut off their cotyledon nodes along the Figure 1 scored part as the explants for induction test;

[0086] Among them, the culture conditions in the MS basal medium are:

[0087] Light intensity is 2000 lx, light time is 12 h / d, and temperature is 25°C.

[0088] S2: Cut off the cotyledon nodes with petioles in a laminar flow hood. The length of the cotyledon nodes with petioles is 5 mm, and place the cotyledon nodes in the callus induction medium obtained by autoclaving at 121°C and 0.01 MPa for 20 min for cultivation to obtain callus;

[0089] Among them, the callus induction medium includes: MS, 3 mg / L TDZ, 0.1 mg / L NAA, 25 g / L sucrose, and 5 g / L agar;

[0090] The culture conditions of the callus induction medium are: light intensity is 2000 lx, light time is 12 h / d, temperature is 25°C, pH is 6.0, and the time is 4 weeks;

[0091] As Figure 2As shown, it is a photo of the callus obtained after culturing in the callus induction medium for 4 weeks.

[0092] S3: The callus cultured for 4 weeks was placed in the adventitious bud induction medium obtained by autoclaving at 121 °C and 0.01 MPa for 20 min for culture to obtain effective buds. The length of the effective buds is 3 cm. As Figure 3 and Figure 4 shown, it is a photo of the obtained effective buds;

[0093] Among them, the adventitious bud induction medium includes: MS, 5 mg / L 6-BA, 0.05 mg / L NAA, 25 g / L sucrose and 5 g / L agar;

[0094] The culture conditions of the adventitious bud induction medium are: light intensity is 2000 lx, light time is 12 h / d, temperature is 25 °C, pH is 6.0, and time is 4 weeks;

[0095] S4: The effective buds were placed in the rooting medium obtained by autoclaving at 121 °C and 0.01 MPa for 20 min for culture to obtain Anthocephalus chinensis seedlings;

[0096] Among them, the rooting medium includes: 1 / 2MS, 0.1 mg / L IBA, 0.05 mg / L NAA, 25 g / L sucrose and 5 g / L agar;

[0097] The culture conditions of the rooting medium are: light intensity is 2000 lx, light time is 12 h / d, temperature is 25 °C, pH is 6.0, and time is 20 days;

[0098] As Figure 5 shown, it is a photo of the rooting of the effective buds obtained after culturing the effective buds in the rooting medium for 20 days;

[0099] As Figure 6 shown, it is a photo of the Anthocephalus chinensis seedlings obtained after culturing the effective buds for 20 days.

[0100] Example 2-9

[0101] The steps and technical parameters in Examples 2-9 are the same as those in Example 1. The difference is that the TDZ and NAA concentrations selected in Examples 2-9 are different from those in Example 1. The specific selected values are shown in Table 1.

[0102] Example 10

[0103] S1: Cultivate aseptic Anthocephalus chinensis elite tree seedlings and select the cotyledon nodes of the seedlings:

[0104] S11: Soak the Anthocephalus chinensis seeds in water for 14 h in a laminar flow hood;

[0105] S12: Transfer the seeds soaked in water to alcohol with a concentration of 75% and soak for 62 s;

[0106] S13: Wash the seeds soaked in alcohol 3 - 5 times with sterile water, and sterilize them with sodium hypochlorite with a concentration of 25% for 7 min to obtain sterilized seeds;

[0107] S14: After washing the sterilized seeds 3 - 5 times with sterile water, transfer the sterilized seeds to the MS basal medium obtained by autoclaving at 120 °C and 0.08 MPa for 25 min for culture to obtain sterile superior Anthocephalus chinensis seedling. After the seedling grows 1 - 2 pairs of true leaves, as Figure 1 shown, cut its cotyledon node along the scribed part as the explant for induction test;

[0108] Among them, the culture conditions in the MS basal medium are:

[0109] The light intensity is 1800 lx, the light time is 14 h / d, and the temperature is 27 °C.

[0110] S2: Cut the cotyledon node with petiole in a laminar flow hood. The length of the cotyledon node with petiole is 5 mm. Place the cotyledon node in the callus induction medium obtained by autoclaving at 120 °C and 0.08 MPa for 25 min for culture to obtain callus;

[0111] Among them, the callus induction medium includes: MS, 5 mg / L TDZ, 0.15 mg / L NAA, 30 g / L sucrose and 7 g / L agar;

[0112] The culture conditions of the callus induction medium are: the light intensity is 1800 lx, the light time is 14 h / d, the temperature is 27 °C, the pH is 5.9, and the time is 4 weeks.

[0113] S3: Place the callus cultured for 4 weeks in the adventitious bud induction medium obtained by autoclaving at 120 °C and 0.08 MPa for 25 min for culture to obtain effective buds, and the length of the effective buds is 2 cm;

[0114] Among them, the adventitious bud induction medium includes: MS, 3 mg / L 6 - BA, 0.03 mg / L NAA, 30 g / L sucrose and 7 g / L agar;

[0115] The culture conditions of the adventitious bud induction medium are: the light intensity is 1800 lx, the light time is 14 h / d, the temperature is 27 °C, the pH is 5.9, and the time is 4 weeks;

[0116] S4: Place the effective buds in a rooting medium obtained by subjecting to high-pressure steam sterilization at 120°C and 0.08 MPa for 25 min for cultivation to obtain Neolamarckia cadamba seedlings;

[0117] Among them, the rooting medium includes: 1 / 2MS, 0.05 mg / L IBA, 0.01 mg / L NAA, 30 g / L sucrose, and 7 g / L agar;

[0118] The cultivation conditions of the rooting medium are: light intensity is 1800 lx, light time is 14 h / d, temperature is 27°C, pH is 5.9, and time is 20 days.

[0119] Example 11

[0120] S1: Cultivate aseptic Neolamarckia cadamba elite tree seedlings and select the cotyledon nodes of the seedlings:

[0121] S11: Soak Neolamarckia cadamba seeds in water for 12 h in a clean bench;

[0122] S12: Transfer the seeds soaked in water to 75% alcohol and soak for 40 s;

[0123] S13: Wash the seeds soaked in alcohol 3 - 5 times with sterile water and sterilize with 25% sodium hypochlorite for 3 min to obtain sterilized seeds;

[0124] S14: After washing the sterilized seeds 3 - 5 times with sterile water, transfer the sterilized seeds to an MS basal medium obtained by subjecting to high-pressure steam sterilization at 125°C and 0.12 MPa for 15 min for cultivation to obtain aseptic Neolamarckia cadamba elite tree seedlings. After the seedlings grow 1 - 2 pairs of true leaves, cut off their cotyledon nodes along the Figure 1 scored part as explants for induction test;

[0125] Among them, the cultivation conditions in the MS basal medium are:

[0126] Light intensity is 2300 lx, light time is 10 h / d, and temperature is 23°C.

[0127] S2: Cut off the cotyledon nodes with petioles in a clean bench. The length of the cotyledon nodes with petioles is 5 mm. Place the cotyledon nodes in a callus induction medium obtained by subjecting to high-pressure steam sterilization at 125°C and 0.12 MPa for 15 min for cultivation to obtain callus;

[0128] Among them, the callus induction medium includes: MS, 5 mg / L TDZ, 0.15 mg / L NAA, 2.7 g / L sucrose, and 6 g / L agar;

[0129] The culture conditions of the callus induction medium are as follows: light intensity is 2300 lx, light time is 10 h / d, temperature is 23 °C, pH is 5.8, and the time is 4 weeks;

[0130] S3: Place the callus cultured for 4 weeks into the adventitious bud induction medium obtained by autoclaving at 125 °C and 0.12 MPa for 15 min for culture to obtain effective buds, and the length of the effective buds is 1.5 cm;

[0131] Among them, the adventitious bud induction medium includes: MS, 7 mg / L 6-BA, 0.07 mg / L NAA, 27 g / L sucrose, and 6 g / L agar;

[0132] The culture conditions of the adventitious bud induction medium are as follows: light intensity is 2300 lx, light time is 10 h / d, temperature is 23 °C, pH is 5.8, and the time is 4 weeks;

[0133] S4: Place the effective buds into the rooting medium obtained by autoclaving at 125 °C and 0.12 MPa for 15 min for culture to obtain Anthocephalus chinensis seedlings;

[0134] Among them, the rooting medium includes: 1 / 2MS, 0.15 mg / L IBA, 0.08 mg / L NAA, 27 g / L sucrose, and 6 g / L agar;

[0135] The culture conditions of the rooting medium are as follows: light intensity is 2300 lx, light time is 10 h / d, temperature is 23 °C, pH is 5.8, and the time is 20 days.

[0136] Example 12

[0137] S1: Culture aseptic Anthocephalus chinensis elite tree seedlings and select the cotyledon nodes of the seedlings:

[0138] S11: Soak Anthocephalus chinensis seeds in water for 12 h in a laminar flow hood;

[0139] S12: Transfer the seeds soaked in water to 75% alcohol and soak for 40 s;

[0140] S13: Wash the seeds soaked in alcohol 3 - 5 times with sterile water and sterilize with 25% sodium hypochlorite for 3 min to obtain sterilized seeds;

[0141] S14: After washing the sterilized seeds 3 - 5 times with sterile water, transfer the sterilized seeds to the MS basal medium obtained by autoclaving at 125°C and 0.12 MPa for 15 min for cultivation to obtain sterile Anthocephalus chinensis superior tree seedlings. After the seedlings grow 1 - 2 pairs of true leaves, cut off their cotyledon nodes along Figure 1 the underlined part in

[0142] as the explants for induction experiments;

[0143] Among them, the cultivation conditions in the MS basal medium are:

[0144] The light intensity is 2300 lx, the light time is 10 h / d, and the temperature is 23°C.

[0145] Among them, the callus induction medium includes: MS, 3 mg / L TDZ, 0.15 mg / L NAA, 27 g / L sucrose and 6 g / L agar;

[0146] The cultivation conditions of the callus induction medium are: the light intensity is 2300 lx, the light time is 10 h / d, the temperature is 23°C, the pH is 5.8, and the time is 4 weeks;

[0147] S3: Place the callus cultivated for 4 weeks in the adventitious bud induction medium obtained by autoclaving at 125°C and 0.12 MPa for 15 min for cultivation to obtain effective buds, and the length of the effective buds is 1.5 cm;

[0148] Among them, the adventitious bud induction medium includes: MS, 7 mg / L 6 - BA, 0.07 mg / L NAA, 27 g / L sucrose and 6 g / L agar;

[0149] The cultivation conditions of the adventitious bud induction medium are: the light intensity is 2300 lx, the light time is 10 h / d, the temperature is 23°C, the pH is 5.8, and the time is 4 weeks;

[0150] S4: Place the effective buds in the rooting medium obtained by autoclaving at 125°C and 0.12 MPa for 15 min for cultivation to obtain Anthocephalus chinensis seedlings;

[0151] Among them, the rooting medium includes: 1 / 2MS, 0.15 mg / L IBA, 0.08 mg / L NAA, 27 g / L sucrose and 6 g / L agar;

[0152] The culture conditions of the rooting medium are as follows: light intensity is 2300 lx, light duration is 10 h / d, temperature is 23 °C, pH is 5.8, and the time is 20 days.

[0153] The callus induction media and culture results of the cotyledon nodes of Melodinus flavus in Examples 1 - 12 are shown in Table 1:

[0154] Table 1 Callus induction media and culture results of the cotyledon nodes of Melodinus flavus in Examples 1 - 12

[0155] Example TDZ (mg / L) NAA (mg / L) Axillary bud germination rate Callus induction rate Callus growth state 1 3 0.1 16.7％ 20％ + 2 5 0.1 26.7％ 13.3％ ++ 3 7 0.1 23.3％ 76.7％ + 4 3 0.15 24.1％ 100％ +++ 5 5 0.15 0％ 100％ ++++ 6 7 0.15 20％ 93.3％ + 7 3 0.2 20％ 90％ +++ 8 5 0.2 19.4％ 67.7％ ++ 9 7 0.2 20％ 80％ + 10 5 0.15 25.2％ 97.7％ +++ 11 5 0.15 19.3％ 98.2％ +++ 12 3 0.15 26.3％ 98.9％ +++

[0156] Note:

[0157] "++++" indicates the best growth state, short callus emergence time, fast growth, large callus mass, smooth surface, dense structure, dark green with slightly pale yellow;

[0158] "+++" indicates good callus growth state, short callus emergence time, relatively fast callus growth, dense structure, green with pale yellow;

[0159] "++" indicates general callus growth state, slow growth, tiny callus mass, white particles attached to the surface, yellowish green;

[0160] "+" indicates poor callus growth state, long callus emergence time, small callus mass, slow growth, white particles attached to the surface, yellowish green.

[0161] As shown in Table 1, the best results are obtained in Examples 4 and 5. However, the axillary bud germination rate in Example 4 is 24.1%, while that in Example 5 is 0%. This shows that TDZ is the best cytokinin for inducing callus from cotyledon node explants. In the MS medium, the MS medium supplemented with 5 mg / L TDZ + 0.15 mg / L NAA has the best callus induction effect, with a callus induction rate of 100% and a dormant axillary bud germination rate of 0%.

[0162] In summary, the present invention provides an efficient regeneration method using the cotyledon nodes of Melodinus flavus as explants. This method is simple, efficient, and can repeatedly achieve in vitro regeneration of cotyledon nodes mediated by callus. According to the study on the influencing factors of plant growth regulators on callus formation, the results show that TDZ is the best cytokinin for inducing callus from cotyledon node explants. In the MS medium, the MS medium supplemented with 5 mg / L TDZ + 0.15 mg / L NAA has the best callus induction effect, with a callus induction rate of 100% and a dormant axillary bud germination rate of 0%. Transferring the induced callus to a medium containing 5 mg / L 6 - BA + 0.05 mg / L NAA for adventitious bud induction, effective buds can be obtained in only 8 weeks, and the average number of effective buds per explant reaches 36.

[0163] The present invention has developed a simple, efficient and reproducible scheme. In this method, the callus induction rate of the cotyledon nodes of Gmelina arborea is as high as 100%, and the physiological state of the callus is good and the budding effect is good. By using the callus of the cotyledon nodes of Gmelina arborea for in vitro regeneration, it can be used for the conservation of Gmelina arborea germplasm resources and large-scale seedling production, and at the same time lays a foundation for transgenic research.

[0164] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those of ordinary skill in the art should understand that they can still modify the technical solutions recorded in the foregoing embodiments, or perform equivalent replacements on some or all of the technical features; and these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. An efficient regeneration method using the leaf nodes of Melia azedarach L. as explants, characterized in that, It includes the following steps: S1: Cultivate aseptic seeds of superior Anthocephalus chinensis trees, and select the cotyledon nodes of the seedling; S2: Place the cotyledon nodes in a callus induction medium for cultivation to obtain callus; S3: Place the callus in a adventitious bud induction medium for cultivation to obtain effective buds; S4: Place the effective buds in a rooting medium for cultivation to obtain Anthocephalus chinensis seedlings; Among them, the callus induction medium includes: MS, 3 - 7 mg / L TDZ, 0.1 - 0.2 mg / L NAA, 25 - 30 g / L sucrose, and 5 - 7 g / L agar.

2. The efficient regeneration method using the Phyllanthus flexuosus leaf node as the explant according to claim 1, characterized in that, The callus induction medium includes: MS, 5 mg / L TDZ, 0.15 mg / L NAA, 25 - 30 g / L sucrose, and 5 - 7 g / L agar.

3. The high-efficiency regeneration method using the leaf nodes of Melodinus xanthocarpus Wall. ex Wight as explants according to claim 1, wherein, The adventitious bud induction medium includes: MS, 3 - 7 mg / L 6 - BA, 0.03 - 0.07 mg / L NAA, 25 - 30 g / L sucrose, and 5 - 7 g / L agar.

4. The efficient regeneration method using the leaf nodes of Melodinus xanthocarpus Wall. ex Wight as explants according to claim 3, wherein, The adventitious bud induction medium includes: MS, 5 mg / L 6 - BA, 0.05 mg / L NAA, 25 - 30 g / L sucrose, and 5 - 7 g / L agar.

5. The efficient regeneration method using the leaf nodes of Melodinus huangliangoensis as explants according to claim 1, wherein, The rooting medium includes: 1 / 2 MS, 0.05 - 0.15 mg / L IBA, 0.01 - 0.08 mg / L NAA, 25 - 30 g / L sucrose, and 5 - 7 g / L agar.

6. The high-efficiency regeneration method using the leaf nodes of Melodinus huangliangoensis as explants according to claim 5, characterized in that, The rooting medium includes: 1 / 2 MS, 0.1 mg / L IBA, 0.05 mg / L NAA, 25 - 30 g / L sucrose, and 5 - 7 g / L agar.

7. The high-efficiency regeneration method using the leaf node of Melodinus officinalis Wall. ex Hook. f. as the explant according to any one of claims 1-6, characterized in that, The MS includes 1900 mg / L potassium nitrate, 332.2 mg / L calcium chloride, 1650 mg / L ammonium nitrate, 180.7 mg / L magnesium sulfate, 170 mg / L potassium dihydrogen phosphate, 0.014 mg / L cobalt chloride, 0.016 mg / L copper sulfate, 6.2 mg / L boric acid, 15.1 mg / L manganese sulfate, 0.21 mg / L sodium molybdate, 4.8 mg / L zinc sulfate, 0.83 mg / L potassium iodide, 27.8 mg / L FeSO4·7H2O, 37.25 mg / L Na2 - EDTA, 2.0 mg / L glycine, 100 mg / L inositol, 0.1 mg / L thiamine VB1, 0.50 mg / L nicotinic acid, 0.5 mg / L pyridoxine VB6.

8. The high-efficiency regeneration method using the leaf nodes of Melodinus flavicarpus as explants according to claim 1, wherein, In step S1, the cultivation of aseptic seeds of superior Anthocephalus chinensis trees includes the following steps: S11: Soak the Anthocephalus chinensis seeds in water for 12 - 24 h; S12: Transfer the seeds soaked in water to 75% - 80% alcohol and soak for 40 - 70 s; S13: Wash the seeds soaked in alcohol with sterile water and sterilize them with 25% - 30% sodium hypochlorite for 3 - 7 min to obtain sterilized seeds; S14: After washing the sterilized seeds with sterile water, transfer the sterilized seeds to an MS basal medium for cultivation to obtain aseptic seeds of superior Anthocephalus chinensis trees.

9. The high-efficiency regeneration method using the leaf nodes of Melodinus huangliangoensis as explants according to claim 8, wherein, In step S14, the cultivation conditions in the MS basal medium are: The light intensity is 1800 - 2300 lx, the light duration is 10 - 14 h / d, and the temperature is 23 - 27 °C.

10. The efficient regeneration method using the leaf node of Melodinus officinalis W. T. Wang as the explant according to claim 1, characterized in that, In step S2, the culture conditions of the callus induction medium are as follows: the light intensity is 1800 - 2300 lx, the light duration is 10 - 14 h / d, the temperature is 23 - 27 °C, the pH is 5.8 - 6.0, and the time is 4 weeks; And / or, the cotyledon node is the cotyledon node with petiole, and the length is 3 - 7 mm.

11. The efficient regeneration method using the leaf node of Melia azedarach L. as the explant according to claim 1, characterized in that, In step S3, the culture conditions of the adventitious bud induction medium are as follows: the light intensity is 1800 - 2300 lx, the light duration is 10 - 14 h / d, the temperature is 23 - 27 °C, the pH is 5.8 - 6.0, and the time is 4 weeks; And / or, the length of the effective bud is greater than 1 cm.

12. The efficient regeneration method using the leaf nodes of Melodinus officinalis Wall. as explants according to claim 1, wherein, In step S4, the culture conditions of the rooting medium are as follows: the light intensity is 1800 - 2300 lx, the light duration is 10 - 14 h / d, the temperature is 23 - 27 °C, the pH is 5.8 - 6.0, and the time is 20 days.

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