Method for embryogenic callus induction of medium-grain coffee

Abstract

The invention relates to the technical field of plant tissue culture, in particular to a medium-grain coffee embryonic callus induction method which comprises the following steps: selecting medium-grain coffee leaves as explants for surface sterilization; sequentially carrying out three culture stages, namely a dedifferentiation stage, an embryogenic induction stage and an embryogenic maintenance stage, on the sterilized explant; in the dedifferentiation stage, a first culture medium added with 2, 4-dichlorphenoxyacetic acid is adopted, and culture is performed under a first culture condition; in the embryogenic induction period, a second culture medium added with 2, 4-dichlorphenoxyacetic acid and 6-benzyladenine is adopted, and culture is performed under a second culture condition; in the embryogenic maintenance period, a third culture medium added with 1-naphthylacetic acid and kinetin is adopted, and culture is performed under a third culture condition; according to the method disclosed by the invention, the compact embryonic callus with low waterlogging degree is obtained, and the induction rate of the embryonic callus reaches 85-95% through three-stage progressive hormone regulation and control, which is obviously higher than that of the prior art by 30-50%.

Description

Technical Field

[0001] The present invention relates to the technical field of plant tissue culture, and particularly relates to a method for inducing embryogenic callus of Coffea canephora Pierre ex A. Froehner. Background Art

[0002] Coffea canephora Pierre ex A. Froehner, also known as Robusta coffee, is one of the most important cash crops in the world, with its cultivation area and yield accounting for more than about 45% of the global coffee industry. Currently, the propagation of Coffea canephora mainly relies on grafting and cutting propagation. These traditional propagation methods have disadvantages such as low propagation coefficient and long growth cycle, and it is difficult to meet the large demand for high-quality seedlings in the modern coffee industry.

[0003] Somatic embryogenesis, as an efficient asexual propagation technology, has important application prospects in the rapid propagation and germplasm resource conservation of Coffea canephora. Among them, the induction of embryogenic callus is a key step in somatic embryogenesis, directly affecting the quantity and quality of subsequent plant regeneration. However, in the existing technology, there are problems such as low induction rate, poor callus quality, and insufficient genetic stability in the induction of embryogenic callus of Coffea canephora.

[0004] The reported techniques for inducing embryogenic callus of Coffea canephora mainly have the following deficiencies: First, most of them adopt a single or dual hormone regulation system, lacking fine regulation of different development stages; Second, the optimization of culture environment parameters is insufficient, resulting in serious waterlogging of callus; Third, there is a lack of a systematic solution for oxidative stress and osmotic pressure regulation, resulting in unstable callus quality. Therefore, there is an urgent need to develop an efficient and stable method for inducing embryogenic callus of Coffea canephora. Summary of the Invention

[0005] In view of the deficiencies of the existing technology, the purpose of the present invention is to provide a method for efficiently inducing embryogenic callus of Coffea canephora. This method uses a three-stage progressive hormone regulation system, combined with an antioxidant protection and osmotic pressure gradient regulation strategy, to significantly improve the induction efficiency and quality of embryogenic callus.

[0006] The purpose of the present invention is to provide a method for inducing embryogenic callus of Coffea canephora, comprising the following steps:

[0007] Select the leaves of Coffea canephora as explants for surface sterilization;

[0008] The sterilized explants are successively passed through three culture stages: dedifferentiation stage, embryogenic induction stage, and embryogenic maintenance stage;

[0009] The dedifferentiation stage uses a first culture medium supplemented with 2,4-dichlorophenoxyacetic acid and is cultured under the first culture conditions;

[0010] During the embryogenic induction period, it is cultured under the second culture conditions using a second culture medium supplemented with 2,4-dichlorophenoxyacetic acid and 6-benzyladenine;

[0011] During the embryogenic maintenance period, it is cultured under the third culture conditions using a third culture medium supplemented with 1-naphthylacetic acid and kinetin;

[0012] Embryogenic callus that is compact and has a low degree of waterlogging is obtained.

[0013] Specifically, the first culture medium comprises the following components:

[0014] A basal medium;

[0015] 2,4-dichlorophenoxyacetic acid 0.8 - 1.2 parts by weight;

[0016] Sucrose 25 - 35 parts by weight;

[0017] L-glutamine 3.5 - 4.5 parts by weight;

[0018] Activated carbon 0.8 - 1.2 parts by weight;

[0019] Agar 6.5 - 7.5 parts by weight.

[0020] Specifically, the second culture medium comprises the following components:

[0021] A basal medium;

[0022] 2,4-dichlorophenoxyacetic acid 0.4 - 0.6 parts by weight;

[0023] 6-benzyladenine 2.0 - 2.5 parts by weight;

[0024] Sucrose 35 - 45 parts by weight;

[0025] Hydrolyzed casein 4.5 - 5.5 parts by weight;

[0026] L-proline 4.5 - 5.5 parts by weight;

[0027] Ascorbic acid 0.9 - 1.1 parts by weight;

[0028] Polyethylene glycol 4000 3.5 - 4.5 parts by weight;

[0029] Phytagel 2.0 - 3.0 parts by weight.

[0030] Specifically, the third culture medium comprises the following components:

[0031] A basal medium;

[0032] 1-naphthylacetic acid 0.2 - 0.3 parts by weight;

[0033] Kinetin 0.9 - 1.1 parts by weight;

[0034] Sucrose 25 - 35 parts by weight;

[0035] Maltose 8 - 12 parts by weight;

[0036] Cystine 0.8 - 1.2 parts by weight;

[0037] Glutathione 0.08 - 0.12 parts by weight;

[0038] Vitamin E 0.008 - 0.012 parts by weight;

[0039] Phytagel 2.0 - 3.0 parts by weight.

[0040] Specifically, the basal medium is a modified MS medium, comprising the following components:

[0041] Ammonium nitrate 15 - 20 parts by weight;

[0042] Potassium nitrate 18 - 22 parts by weight;

[0043] Magnesium sulfate heptahydrate 3.5 - 4.5 parts by weight;

[0044] Potassium dihydrogen phosphate 1.5 - 2.5 parts by weight;

[0045] Calcium chloride dihydrate 3.0 - 4.0 parts by weight;

[0046] MS trace elements;

[0047] Iron source;

[0048] Organic components;

[0049] The MS trace elements include: Manganese sulfate monohydrate 1.5 - 2.2 parts by weight, Zinc sulfate heptahydrate 0.8 - 1.2 parts by weight, Boric acid 0.5 - 0.7 parts by weight, Potassium iodide 0.07 - 0.09 parts by weight, Sodium molybdate dihydrate 0.02 - 0.03 parts by weight, Copper sulfate pentahydrate 0.002 - 0.003 parts by weight, Cobalt chloride hexahydrate 0.002 - 0.003 parts by weight; The iron source includes: EDTA-Na 2 3.5 - 4.5 parts by weight, Ferrous sulfate heptahydrate 2.5 - 3.5 parts by weight; The organic components include: Inositol 9 - 11 parts by weight, Thiamine hydrochloride 0.09 - 0.11 parts by weight, Pyridoxine hydrochloride 0.045 - 0.055 parts by weight, Nicotinic acid 0.045 - 0.055 parts by weight, Glycine 0.18 - 0.22 parts by weight.

[0050] Specifically, the basal medium further comprises the following trace element enhancers:

[0051] Sodium selenate 0.0008 - 0.0012 parts by weight;

[0052] Nickel chloride hexahydrate 0.0003 - 0.0007 parts by weight;

[0053] Sodium silicate nonahydrate 0.08 - 0.12 parts by weight.

[0054] Specifically, the first culture conditions are: temperature 25 ± 1 °C, completely dark culture for 7 - 10 days and then changed to a 16 / 8 hour light / dark cycle, light intensity 20 - 30 μmol·m-2·s-1, pH value 5.6 - 5.8, culture for 21 - 28 days.

[0055] Specifically, the second culture conditions are: temperature 26 ± 1 °C, 12 / 12 hour light / dark cycle, light intensity 40 - 50 μmol·m-2·s-1, pH value 5.4 - 5.6, culture for 14 - 21 days.

[0056] Specifically, the third culture conditions are: temperature 24 ± 1 °C, 16 / 8 hour light / dark cycle, light intensity 50 - 60 μmol·m-2·s-1, pH value 5.2 - 5.4, culture for 21 - 28 days.

[0057] Specifically, the steps of surface sterilization include: soaking in 75% ethanol for 30 - 40 seconds; soaking in 0.1% mercuric chloride solution for 7 - 9 minutes; treating with 0.5 - 1.0% sodium hypochlorite solution for 10 - 12 minutes; washing with sterile water 5 - 6 times, 3 - 5 minutes each time.

[0058] Compared with the prior art, the present invention has the following beneficial effects:

[0059] 1. Through three-stage progressive hormone regulation, the induction rate of embryogenic callus reaches 85 - 95%, significantly higher than 30 - 50% of the prior art;

[0060] 2. Adopting a synergistic mechanism of antioxidant protection and osmotic pressure gradient regulation, the induced embryogenic callus has excellent characteristics such as being compact, having a low degree of waterlogging, and high embryogenic potential;

[0061] 3. Through nitrogen source balance and synergistic effect of trace elements, the cultured embryogenic callus can be stably subcultured 6 - 8 times without losing its embryogenicity, providing high-quality starting materials for large-scale asexual propagation of Coffea canephora Pierre ex A. Froehner;

[0062] 4. The optimized precise control scheme of physical environmental factors improves the controllability and repeatability of the culture process, laying a foundation for large-scale production of embryogenic callus of Coffea canephora Pierre ex A. Froehner. Detailed implementation mode

[0063] The present invention will be further described in detail below in conjunction with specific embodiments, but the protection scope of the present invention is not limited thereto.

[0064] The present invention provides a method for inducing embryogenic callus of Coffea canephora Pierre ex A. Froehner, comprising the following steps: selecting Coffea canephora Pierre ex A. Froehner leaves as explants for surface sterilization; subjecting the sterilized explants to three culture stages in sequence: dedifferentiation stage, embryogenic induction stage and embryogenic maintenance stage; using a first culture medium supplemented with 2,4-dichlorophenoxyacetic acid for culturing under a first culture condition during the dedifferentiation stage; using a second culture medium supplemented with 2,4-dichlorophenoxyacetic acid and 6-benzyladenine for culturing under a second culture condition during the embryogenic induction stage; using a third culture medium supplemented with 1-naphthylacetic acid and kinetin for culturing under a third culture condition during the embryogenic maintenance stage; and obtaining compact embryogenic callus with low degree of waterlogging.

[0065] The present invention adopts a three-stage progressive hormone regulation system, scientifically divides the process of inducing embryogenic callus into three stages: dedifferentiation stage, embryogenic induction stage and embryogenic maintenance stage, and designs specific culture medium formulations and culture conditions for each stage. Among them, the main goal of the dedifferentiation stage is to promote cell dedifferentiation with high-concentration 2,4-D; the embryogenic induction stage induces embryogenic potential through an optimized cytokinin / auxin ratio (about 4:1); and the embryogenic maintenance stage uses a combination of NAA and KT to maintain the stability of the embryogenic state. The orderly connection of the three stages ensures the efficient transformation from ordinary somatic cells to embryogenic cells.

[0066] Meanwhile, the synergistic mechanism of antioxidant protection and osmotic pressure gradient regulation in the present invention solves the problems of serious waterlogging and unstable embryogenicity of callus in traditional methods. By adding specific antioxidants (such as activated carbon, ascorbic acid, glutathione and vitamin E) at different stages, a multi-level antioxidant defense system is constructed; combined with the carbon source gradient regulation strategy (phased adjustment of sucrose concentration and introduction of maltose), an osmotic environment conducive to embryogenic development is created.

[0067] To further optimize the culture effect, the present invention also adopts a strategy of nitrogen source balance and synergistic enhancement of trace elements. By reasonably matching organic nitrogen sources such as L-glutamine, casein hydrolysate, L-proline and cystine, the nitrogen utilization efficiency is improved; at the same time, trace element enhancers such as selenium, nickel and silicon are introduced to promote the activation of specific metabolic pathways.

[0068] The compounds used in the present invention are summarized as follows: ammonium nitrate (NH 4 NO 3 ), potassium nitrate (KNO 3 ), magnesium sulfate heptahydrate (MgSO 4 ·7H 2 O), potassium dihydrogen phosphate (KH 2 PO 4), calcium chloride dihydrate (CaCl 2 ·2H 2 O), manganese sulfate monohydrate (MnSO 4 ·H 2 O), zinc sulfate heptahydrate (ZnSO 4 ·7H 2 O), boric acid (H 3 BO 3 ), potassium iodide (KI), sodium molybdate dihydrate (Na 2 MoO 4 ·2H 2 O), copper sulfate pentahydrate (CuSO 4 ·5H 2 O), cobalt chloride hexahydrate (CoCl 2 ·6H 2 O), EDTA-Na 2 , ferrous sulfate heptahydrate (FeSO 4 ·7H 2 O), inositol (C 6 H 12 O 6 ), thiamine hydrochloride (vitamin B 1 ), pyridoxine hydrochloride (vitamin B 6 ), nicotinic acid, glycine (C 2 H 5 NO 2 ), 2,4-dichlorophenoxyacetic acid (2,4-D), sucrose (C 12 H 22 O 11 ), L-glutamine (C 5 H 10 N 2 O 3 ), activated carbon (Sigma-Aldrich, C9157), agar (Sigma-Aldrich, A1296), 6-benzyladenine (BA or 6-BA), casein hydrolysate (Sigma-Aldrich, C7290), L-proline (C 5 H 9 NO 2 ), ascorbic acid (vitamin C), polyethylene glycol 4000 (PEG 4000), Phytagel (Phytagel, Sigma-Aldrich, P8169), 1-naphthaleneacetic acid (NAA), kinetin (KT or Kinetin), maltose (C 12 H 22 O 11 ), cystine (C 6 H 12 N 2 O4 S 2 ) glutathione (C 10 H 17 N 3 O 6 S), vitamin E (tocopheryl acetate), sodium selenate (Na 2 SeO 4 ), nickel chloride hexahydrate (NiCl 2 ·6H 2 O), sodium silicate nonahydrate (Na 2 SiO 3 ·9H 2 O), mercuric chloride (HgCl 2 ), sodium hypochlorite (NaClO), ethanol, thiobarbituric acid (TBA).

[0069] Example 1

[0070] A method for inducing embryogenic callus of Coffea canephora, comprising the following steps:

[0071] (1) Selection and sterilization of explants: Select healthy plants of Coffea canephora Pierre ex A. Froehner variety that have grown for 3 months, take the second pair of fully expanded leaves, and cut them into square segments of 1.5 cm 2 . Use the following sterilization protocol: soak in 75% ethanol for 30 seconds, soak in 0.1% mercuric chloride solution for 7 minutes, treat with 0.5% sodium hypochlorite solution for 10 minutes, and wash with sterile water 5 times, 3 minutes each time.

[0072] (2) Culture in the dedifferentiation stage: Inoculate the sterilized explants with the cut edge facing down on the first medium. The first medium includes the following components: basal medium; 2,4-dichlorophenoxyacetic acid 0.8 parts by weight; sucrose 25 parts by weight; L-glutamine 3.5 parts by weight; activated carbon 0.8 parts by weight; agar 6.5 parts by weight. Among them, the basal medium is a modified MS medium, including the following components: ammonium nitrate 15 parts by weight; potassium nitrate 18 parts by weight; magnesium sulfate heptahydrate 3.5 parts by weight; potassium dihydrogen phosphate 1.5 parts by weight; calcium chloride dihydrate 3.0 parts by weight; MS trace elements; iron source; organic components; MS trace elements include: manganese sulfate monohydrate 1.5 parts by weight, zinc sulfate heptahydrate 0.8 parts by weight, boric acid 0.5 parts by weight, potassium iodide 0.07 parts by weight, sodium molybdate dihydrate 0.02 parts by weight, copper sulfate pentahydrate 0.002 parts by weight, cobalt chloride hexahydrate 0.002 parts by weight;

[0073] The iron source includes: EDTA-Na 2 3.5 parts by weight, ferrous sulfate heptahydrate 2.5 parts by weight;

[0074] The organic components include: 9 parts by weight of inositol, 0.09 parts by weight of thiamine hydrochloride, 0.045 parts by weight of pyridoxine hydrochloride, 0.045 parts by weight of nicotinic acid, and 0.18 parts by weight of glycine.

[0075] The first culture conditions are: temperature 25°C, culturing in complete darkness for 7 days and then changing to a 16 / 8 hour light / dark cycle, light intensity 20 μmol·m-2·s-1, pH value 5.6, and culturing for 21 days.

[0076] At this stage, 2,4-dichlorophenoxyacetic acid, as a potent auxin, can break the cell differentiation state and promote cell dedifferentiation and division. A lower concentration of sucrose (25 parts by weight) provides a suitable carbon source and osmotic pressure environment for cell division in the initial stage. L-glutamine, as an easily utilizable nitrogen source, accelerates cell metabolism and protein synthesis. The addition of activated carbon can adsorb harmful phenolic substances and peroxides generated during the culturing process, reducing oxidative stress. After 21 days of culturing, milky white dense callus can be observed to form at the cut of the explant.

[0077] (3) Culturing in the embryogenic induction stage: Transfer the formed callus to the second culture medium. The second culture medium includes the following components: basal medium; 0.4 parts by weight of 2,4-dichlorophenoxyacetic acid; 2.0 parts by weight of 6-benzyladenine; 35 parts by weight of sucrose; 4.5 parts by weight of hydrolyzed casein; 4.5 parts by weight of L-proline; 0.9 parts by weight of ascorbic acid; 3.5 parts by weight of polyethylene glycol 4000; 2.0 parts by weight of phytagel. The second culture conditions are: temperature 26°C, 12 / 12 hour light / dark cycle, light intensity 40 μmol·m-2·s-1, pH value 5.4, and culturing for 14 days.

[0078] At this stage, the concentration of 2,4-dichlorophenoxyacetic acid decreases (0.4 parts by weight) while the concentration of 6-benzyladenine increases (2.0 parts by weight), making the cytokinin / auxin ratio approximately 4:1, and this ratio has been proven to be beneficial for the expression of genes related to somatic embryogenesis. The increase in sucrose concentration (35 parts by weight) creates a slight osmotic stress environment and promotes embryogenic differentiation. The addition of hydrolyzed casein and L-proline provides diverse amino acids, and at the same time, L-proline also acts as an osmotic regulator. Ascorbic acid, as an antioxidant that directly scavenges free radicals, protects cells from oxidative damage. Polyethylene glycol 4000 can promote signal communication between cells while maintaining the osmotic pressure. After 14 days of culturing, yellowish-white granular structures can be observed to form on the surface of the callus, which are the preliminary characteristics of embryogenic development.

[0079] (4) Embryogenic maintenance culture: Transfer the callus containing granular structures to the third medium. The third medium comprises the following components: basal medium; 1-naphthaleneacetic acid 0.2 parts by weight; kinetin 0.9 parts by weight; sucrose 25 parts by weight; maltose 8 parts by weight; cystine 0.8 parts by weight; glutathione 0.08 parts by weight; vitamin E 0.008 parts by weight; phytagel 2.0 parts by weight. The third culture conditions are: temperature 24 °C, 16 / 8 hour light / dark cycle, light intensity 50 μmol·m-2·s-1, pH value 5.2, culture for 21 days.

[0080] During the embryogenic maintenance stage, the combination of 1-naphthaleneacetic acid and kinetin can stabilize the embryogenic state and prevent early differentiation. The reduced sucrose concentration (25 parts by weight) alleviates osmotic stress, while the introduction of maltose (8 parts by weight) provides a more stable carbon source. Cystine promotes sulfur metabolism and protein synthesis, and the combination of glutathione and vitamin E, as potent antioxidants, constructs a complete antioxidant defense system. After 21 days of culture, high-quality embryogenic callus is obtained, showing milky white to light yellow, highly dense, smooth surface, low degree of waterlogging, and obvious embryogenic characteristics.

[0081] In this example, the induction rate of embryogenic callus reaches 86%, the quality of the callus is excellent, and the subsequent somatic embryo differentiation rate reaches 74%, far higher than 30 - 40% of the traditional method.

[0082] Example 2

[0083] A method for inducing embryogenic callus of Coffea canephora, comprising the following steps:

[0084] (1) Explant selection and sterilization: Select healthy plants of Coffea canephora Pierre ex A. Froehner variety that have grown for 4 months, take the third pair of fully expanded leaves, and cut them into square segments of 2.0 cm 2 . Adopt the following sterilization protocol: soak in 75% ethanol for 40 seconds, soak in 0.1% mercuric chloride solution for 9 minutes, treat with 1.0% sodium hypochlorite solution for 12 minutes, and wash with sterile water 6 times, 5 minutes each time.

[0085] (2) Dedifferentiation culture: Inoculate the sterilized explants on the first medium. The first medium comprises the following components: basal medium; 2,4-dichlorophenoxyacetic acid 1.2 parts by weight; sucrose 35 parts by weight; L-glutamine 4.5 parts by weight; activated carbon 1.2 parts by weight; agar 7.5 parts by weight. Among them, the basal medium comprises the following components: ammonium nitrate 20 parts by weight; potassium nitrate 22 parts by weight; magnesium sulfate heptahydrate 4.5 parts by weight; potassium dihydrogen phosphate 2.5 parts by weight; calcium chloride dihydrate 4.0 parts by weight; MS trace elements; iron source; organic components;

[0086] The MS trace elements include: 2.2 parts by weight of manganese sulfate monohydrate, 1.2 parts by weight of zinc sulfate heptahydrate, 0.7 part by weight of boric acid, 0.09 part by weight of potassium iodide, 0.03 part by weight of sodium molybdate dihydrate, 0.003 part by weight of copper sulfate pentahydrate, 0.003 part by weight of cobalt chloride hexahydrate;

[0087] The iron source includes: EDTA-Na 2 4.5 parts by weight, 3.5 parts by weight of ferrous sulfate heptahydrate;

[0088] The organic components include: 11 parts by weight of inositol, 0.11 part by weight of thiamine hydrochloride, 0.055 part by weight of pyridoxine hydrochloride, 0.055 part by weight of nicotinic acid, 0.22 part by weight of glycine;

[0089] and 0.0012 part by weight of sodium selenate; 0.0007 part by weight of nickel chloride hexahydrate; 0.12 part by weight of sodium silicate nonahydrate. The first culture conditions are: temperature 25°C, culturing in complete darkness for 10 days and then changing to a 16 / 8 hour light / dark cycle, light intensity 30 μmol·m-2·s-1, pH value 5.8, culturing for 28 days.

[0090] (3) Culturing in the embryogenic induction stage: Transfer the formed callus to the second culture medium. The second culture medium includes the following components: basic medium; 0.6 part by weight of 2,4-dichlorophenoxyacetic acid; 2.5 parts by weight of 6-benzyladenine; 45 parts by weight of sucrose; 5.5 parts by weight of hydrolyzed casein; 5.5 parts by weight of L-proline; 1.1 parts by weight of ascorbic acid; 4.5 parts by weight of polyethylene glycol 4000; 3.0 parts by weight of phytagel. The second culture conditions are: temperature 26°C, 12 / 12 hour light / dark cycle, light intensity 50 μmol·m-2·s-1, pH value 5.6, culturing for 21 days.

[0091] (4) Culturing in the embryogenic maintenance stage: Transfer the callus containing granular structures to the third culture medium. The third culture medium includes the following components: basic medium; 0.3 part by weight of 1-naphthylacetic acid; 1.1 part by weight of kinetin; 35 parts by weight of sucrose; 12 parts by weight of maltose; 1.2 parts by weight of cystine; 0.12 part by weight of glutathione; 0.012 part by weight of vitamin E; 3.0 parts by weight of phytagel. The third culture conditions are: temperature 24°C, 16 / 8 hour light / dark cycle, light intensity 60 μmol·m-2·s-1, pH value 5.4, culturing for 28 days.

[0092] In this example, the upper limit values of each component were adopted, and trace element enhancers were added. By increasing the concentration of 2,4-D (1.2 parts by weight) and prolonging the dark culture time (10 days), the dedifferentiation effect was enhanced; by increasing the concentration of cytokinin (6-BA 2.5 parts by weight) and sucrose concentration (45 parts by weight), the embryogenic induction effect was enhanced; by increasing the concentration of antioxidants (glutathione 0.12 parts by weight, vitamin E 0.012 parts by weight), the cell protection effect during the embryogenic maintenance stage was enhanced. This example is particularly suitable for coffee varieties with poor induction response, and the induction rate of embryogenic callus reaches 90%, and the callus can be stably subcultured more than 8 times.

[0093] Example 3

[0094] A method for inducing embryogenic callus of Coffea canephora, comprising the following steps:

[0095] (1) Selection and sterilization of explants: Select healthy plants of Coffea canephora Pierre ex A. Froehner variety that have grown for 3.5 months, take the second to third pairs of fully expanded leaves, and cut them into square segments of 1.8 cm 2 . The following sterilization protocol is adopted: soak in 75% ethanol for 35 seconds, soak in 0.1% mercuric chloride solution for 8 minutes, treat with 0.8% sodium hypochlorite solution for 11 minutes, and wash with sterile water 5 times, 4 minutes each time.

[0096] (2) Culture during the dedifferentiation period: Inoculate the sterilized explants on the first medium. The first medium includes the following components: basal medium; 2,4-dichlorophenoxyacetic acid 1.0 part by weight; sucrose 30 parts by weight; L-glutamine 4.0 parts by weight; activated carbon 1.0 part by weight; agar 7.0 parts by weight. Among them, the basal medium includes the following components: ammonium nitrate 16.5 parts by weight; potassium nitrate 19 parts by weight; magnesium sulfate heptahydrate 3.7 parts by weight; potassium dihydrogen phosphate 1.7 parts by weight; calcium chloride dihydrate 3.3 parts by weight; MS trace elements; iron source; organic components; and sodium selenate 0.001 part by weight; nickel chloride hexahydrate 0.0005 part by weight; sodium silicate nonahydrate 0.1 part by weight.

[0097] MS trace elements include: manganese sulfate monohydrate 1.85 parts by weight, zinc sulfate heptahydrate 1.0 part by weight, boric acid 0.6 part by weight, potassium iodide 0.08 part by weight, sodium molybdate dihydrate 0.025 part by weight, copper sulfate pentahydrate 0.0025 part by weight, cobalt chloride hexahydrate 0.0025 part by weight;

[0098] The iron source includes: EDTA-Na 2 4.0 parts by weight, ferrous sulfate heptahydrate 3.0 parts by weight;

[0099] The organic components include: 10 parts by weight of inositol, 0.1 part by weight of thiamine hydrochloride, 0.05 part by weight of pyridoxine hydrochloride, 0.05 part by weight of nicotinic acid, and 0.2 part by weight of glycine;

[0100] The first culture conditions are as follows: temperature 25°C, cultured in complete darkness for 8 days and then transferred to a 16 / 8 hour light / dark cycle, light intensity 25 μmol·m-2·s-1, pH value 5.7, cultured for 24 days.

[0101] (3) Culture in the embryogenic induction stage: Transfer the formed callus to the second culture medium. The second culture medium includes the following components: basal medium; 0.5 part by weight of 2,4-dichlorophenoxyacetic acid; 2.2 parts by weight of 6-benzyladenine; 40 parts by weight of sucrose; 5.0 parts by weight of hydrolyzed casein; 5.0 parts by weight of L-proline; 1.0 part by weight of ascorbic acid; 4.0 parts by weight of polyethylene glycol 4000; 2.5 parts by weight of phytagel. The second culture conditions are as follows: temperature 26°C, 12 / 12 hour light / dark cycle, light intensity 45 μmol·m-2·s-1, pH value 5.5, cultured for 18 days.

[0102] (4) Culture in the embryogenic maintenance stage: Transfer the callus containing granular structures to the third culture medium. The third culture medium includes the following components: basal medium; 0.25 part by weight of 1-naphthylacetic acid; 1.0 part by weight of kinetin; 30 parts by weight of sucrose; 10 parts by weight of maltose; 1.0 part by weight of cystine; 0.1 part by weight of glutathione; 0.01 part by weight of vitamin E; 2.5 parts by weight of phytagel. The third culture conditions are as follows: temperature 24°C, 16 / 8 hour light / dark cycle, light intensity 55 μmol·m-2·s-1, pH value 5.3, cultured for 25 days.

[0103] This example uses the intermediate values of each parameter, representing the optimal combination scheme of the present invention. The balanced ratio of 2,4-D (1.0 part by weight) and sucrose (30 parts by weight) creates a suitable dedifferentiation environment; the approximate 4:1 ratio of 2,4-D (0.5 part by weight) and 6-BA (2.2 parts by weight) optimizes the embryogenic induction effect; the combination of NAA (0.25 part by weight) and KT (1.0 part by weight), as well as the synergistic effect of glutathione (0.1 part by weight) and vitamin E (0.01 part by weight), form a complete embryogenic maintenance and protection system. Under this scheme, the induction rate of embryogenic callus reaches 95%, the embryogenic quality is the best, showing a highly dense light yellow tissue, completely non-waterlogged, and the embryogenic stability is the highest.

[0104] Example 4

[0105] A method for inducing embryogenic callus of Coffea canephora Pierre var. robusta includes the following steps:

[0106] (1) Explant selection and sterilization: Select healthy plants of the Coffea canephora Pierre ex A. Froehner variety that have grown for 3.5 months, take the third pair of fully expanded leaves, and cut them into square segments of 1.7 cm 2 . The following sterilization protocol is adopted: soak in 75% ethanol for 35 seconds, soak in 0.1% mercuric chloride solution for 8 minutes, treat with 0.7% sodium hypochlorite solution for 11 minutes, and wash with sterile water 5 times, 4 minutes each time.

[0107] (2) Cultivation in the dedifferentiation stage: Inoculate the sterilized explants onto the first medium. The first medium includes the following components: basal medium; 1.0 part by weight of 2,4-dichlorophenoxyacetic acid; 25 parts by weight of sucrose; 4.0 parts by weight of L-glutamine; 1.0 part by weight of activated carbon; 7.0 parts by weight of agar. Among them, the basal medium includes the following components: 17 parts by weight of ammonium nitrate; 20 parts by weight of potassium nitrate; 4.0 parts by weight of magnesium sulfate heptahydrate; 2.0 parts by weight of potassium dihydrogen phosphate; 3.5 parts by weight of calcium chloride dihydrate; MS trace elements; iron source; organic components; and 0.001 part by weight of sodium selenate; 0.0005 part by weight of nickel chloride hexahydrate; 0.1 part by weight of sodium silicate nonahydrate.

[0108] The MS trace elements include: 1.7 parts by weight of manganese sulfate monohydrate, 0.9 parts by weight of zinc sulfate heptahydrate, 0.55 parts by weight of boric acid, 0.075 parts by weight of potassium iodide, 0.022 parts by weight of sodium molybdate dihydrate, 0.0022 parts by weight of copper sulfate pentahydrate, 0.0022 parts by weight of cobalt chloride hexahydrate;

[0109] The iron source includes: EDTA-Na 2 3.7 parts by weight, 2.7 parts by weight of ferrous sulfate heptahydrate;

[0110] The organic components include: 9.5 parts by weight of inositol, 0.095 parts by weight of thiamine hydrochloride, 0.048 parts by weight of pyridoxine hydrochloride, 0.048 parts by weight of nicotinic acid, 0.19 parts by weight of glycine;

[0111] The first culture conditions are: temperature 24°C, complete darkness for 9 days and then change to a 16 / 8 hour light / dark cycle, light intensity 25 μmol·m-2·s-1, pH value 5.7, culture for 25 days.

[0112] In this example, the temperature (24°C) and sucrose concentration (25 parts by weight) in the dedifferentiation stage are particularly reduced, considering that the Coffea canephora Pierre ex A. Froehner variety has better dedifferentiation effect at lower temperatures.

[0113] (3) Embryogenic induction stage culture: Transfer the formed callus to the second medium. The second medium comprises the following components: basal medium; 2,4-dichlorophenoxyacetic acid 0.4 parts by weight; 6-benzyladenine 2.2 parts by weight; sucrose 40 parts by weight; casein hydrolysate 5.0 parts by weight; L-proline 5.0 parts by weight; ascorbic acid 1.0 part by weight; polyethylene glycol 4000 4.0 parts by weight; phytagel 2.5 parts by weight. The second culture conditions are: temperature 27 °C, 12 / 12 h light / dark cycle, light intensity 45 μmol·m-2·s-1, pH value 5.5, culture for 18 days.

[0114] In this example, the temperature (27 °C) during the embryogenic induction stage was particularly increased because the (C.canephora Pierre ex A.Froehner) variety requires a higher temperature to activate the relevant metabolic pathways during the embryogenic induction stage.

[0115] (4) Embryogenic maintenance stage culture: Transfer the callus containing granular structures to the third medium. The third medium comprises the following components: basal medium; 1-naphthaleneacetic acid 0.25 parts by weight; kinetin 1.0 part by weight; sucrose 30 parts by weight; maltose 10 parts by weight; cystine 1.0 part by weight; glutathione 0.1 part by weight; vitamin E 0.01 part by weight; phytagel 2.5 parts by weight. The third culture conditions are: temperature 24 °C, 16 / 8 h light / dark cycle, light intensity 55 μmol·m-2·s-1, pH value 5.3, culture for 25 days.

[0116] This example particularly optimized and adjusted the temperature parameters according to the physiological characteristics of the C.canephora Pierre ex A.Froehner variety. The induction rate of embryogenic callus reached 88%, and the quality of the callus was good, which was suitable for the construction of the somatic embryogenesis system of this variety.

[0117] Example 5

[0118] A method for inducing embryogenic callus of Coffea canephora, comprising the following steps:

[0119] (1) Explant selection and sterilization: Select healthy plants of the Coffea canephora (C.canephora Pierre ex A.Froehner) variety that have grown for 4 months, take the second pair of fully expanded leaves, and cut them into square segments of 1.6 cm 2 in size. Adopt the following sterilization scheme: soak in 75% ethanol for 32 seconds, soak in 0.1% mercuric chloride solution for 8 minutes, treat with 0.6% sodium hypochlorite solution for 10 minutes, and wash with sterile water 5 times, 3 minutes each time.

[0120] (2) Dedifferentiation stage culture: The sterilized explants are inoculated on the first medium. The first medium comprises the following components: a basal medium; 0.9 parts by weight of 2,4-dichlorophenoxyacetic acid; 28 parts by weight of sucrose; 4.0 parts by weight of L-glutamine; 1.0 part by weight of activated carbon; 7.0 parts by weight of agar. Among them, the basal medium comprises the following components: 18 parts by weight of ammonium nitrate; 20 parts by weight of potassium nitrate; 4.0 parts by weight of magnesium sulfate heptahydrate; 2.0 parts by weight of potassium dihydrogen phosphate; 3.5 parts by weight of calcium chloride dihydrate; MS trace elements; an iron source; organic components; and 0.001 part by weight of sodium selenate; 0.0005 part by weight of nickel chloride hexahydrate; 0.1 part by weight of sodium silicate nonahydrate.

[0121] The MS trace elements include: 2.0 parts by weight of manganese sulfate monohydrate, 1.1 parts by weight of zinc sulfate heptahydrate, 0.65 part by weight of boric acid, 0.085 part by weight of potassium iodide, 0.028 part by weight of sodium molybdate dihydrate, 0.0028 part by weight of copper sulfate pentahydrate, 0.0028 part by weight of cobalt chloride hexahydrate;

[0122] The iron source includes: EDTA-Na 2 4.3 parts by weight, 3.3 parts by weight of ferrous sulfate heptahydrate;

[0123] The organic components include: 10.5 parts by weight of inositol, 0.105 part by weight of thiamine hydrochloride, 0.052 part by weight of pyridoxine hydrochloride, 0.052 part by weight of nicotinic acid, 0.21 part by weight of glycine;

[0124] The first culture conditions are: temperature 25°C, complete darkness for 8 days and then changed to a 16 / 8 hour light / dark cycle, light intensity 22 μmol·m-2·s-1, pH value 5.7, cultured for 24 days.

[0125] (3) Embryogenic induction stage culture: The formed callus is transferred to the second medium. The second medium comprises the following components: a basal medium; 0.45 part by weight of 2,4-dichlorophenoxyacetic acid; 2.1 parts by weight of 6-benzyladenine; 38 parts by weight of sucrose; 4.8 parts by weight of hydrolyzed casein; 4.8 parts by weight of L-proline; 1.0 part by weight of ascorbic acid; 3.8 parts by weight of polyethylene glycol 4000; 2.4 parts by weight of phytagel. The second culture conditions are: temperature 26°C, 12 / 12 hour light / dark cycle, light intensity 42 μmol·m-2·s-1, pH value 5.5, cultured for 16 days.

[0126] (4) Embryogenic maintenance culture: Transfer the callus containing granular structures to the third culture medium. The third culture medium comprises the following components: a basal medium; 0.22 parts by weight of 1-naphthaleneacetic acid; 0.95 parts by weight of kinetin; 28 parts by weight of sucrose; 9 parts by weight of maltose; 0.9 parts by weight of cystine; 0.09 parts by weight of glutathione; 0.009 parts by weight of vitamin E; 2.3 parts by weight of phytagel. The third culture conditions are as follows: temperature 24 °C, 16 / 8 hour light / dark cycle, light intensity 52 μmol·m-2·s-1, pH value 5.3, and culture for 22 days.

[0127] In this example, according to the resistance characteristics of the Coffea canephora Pierre ex A. Froehner variety, the concentrations of some components were appropriately reduced. As a rust-resistant variety, Coffea canephora Pierre ex A. Froehner has a relatively thick cell wall and relatively low embryogenic development potential. By finely adjusting the hormone concentration and culture time, the induction rate of embryogenic callus was successfully increased to 82%, providing a reliable method for somatic embryogenesis of this disease-resistant variety.

[0128] The above examples prove that the method for inducing embryogenic callus of Coffea canephora Pierre ex A. Froehner provided by the present invention is efficient, stable, and highly adaptable, and can be appropriately adjusted according to the physiological characteristics of different varieties to achieve efficient induction of high-quality embryogenic callus. The present invention solves the problems in the prior art such as low induction rate of embryogenic callus of Coffea canephora Pierre ex A. Froehner, poor quality of callus, and insufficient genetic stability, and provides key technical support for somatic embryogenesis and large-scale asexual propagation of Coffea canephora Pierre ex A. Froehner.

[0129] The above is only a preferred embodiment of the present invention and is not intended to limit the present invention. The protection scope of the present invention shall be subject to the claims.

[0130] In order to verify the superiority of the three-stage progressive hormone regulation system combined with antioxidant protection and osmotic pressure gradient regulation strategy proposed by the present invention, the following comparative experiments were designed.

[0131] Comparative Example 1: Traditional single hormone system

[0132] In this comparative example, a traditional single hormone system was used to induce embryogenic callus of Coffea canephora Pierre ex A. Froehner. The specific method is as follows:

[0133] Select the leaves of Coffea canephora Pierre ex A. Froehner variety as explants. After being treated by a standard sterilization procedure, they are inoculated on an MS medium containing 1.0 part by weight of 2,4-D and 0.5 part by weight of BAP. The medium also contains 30 parts by weight of sucrose and 7.0 parts by weight of agar, and the pH value is adjusted to 5.7. The culture conditions are temperature 25 °C, 16 / 8 hour (light / dark) light cycle, and light intensity 30 μmol·m-2·s-1. After culturing for 60 days, observe the formation of callus.

[0134] Comparative Example 2: Two-stage culture system

[0135] In this comparative example, a simplified two-stage culture system was used for the induction of embryogenic callus of Coffea canephora. In the first stage, high-concentration 2,4-D was used for dedifferentiation induction, and in the second stage, cytokinin was directly used to maintain embryogenicity, omitting the embryogenic induction period in the present invention. The specific method is as follows:

[0136] Dedifferentiation period: The sterilized leaves of Coffea canephora Pierre ex A. Froehner variety were inoculated on an MS medium containing 1.0 part by weight of 2,4-D, 30 parts by weight of sucrose, and 7.0 parts by weight of agar, with a pH value of 5.7. The culture conditions were a temperature of 25°C, a 16 / 8 hour (light / dark) photoperiod after 8 days of complete darkness, a light intensity of 25 μmol·m-2·s-1, and cultured for 30 days.

[0137] Embryogenic maintenance period: The formed callus was transferred to an MS medium containing 1.0 part by weight of KT, 0.25 part by weight of NAA, 30 parts by weight of sucrose, and 2.5 parts by weight of phytagel, with a pH value of 5.3. The culture conditions were a temperature of 24°C, a 16 / 8 hour (light / dark) photoperiod, a light intensity of 55 μmol·m-2·s-1, and cultured for 30 days.

[0138] Comparative Example 3: Lack of antioxidant protection system

[0139] In this comparative example, the three-stage progressive hormone regulation system of the present invention was used, but antioxidants (including activated carbon, ascorbic acid, glutathione, and vitamin E) were not added in each stage to verify the importance of the antioxidant protection system. Other culture conditions were the same as those in Example 3.

[0140] Comparative Example 4: Lack of osmotic pressure gradient regulation

[0141] In this comparative example, the three-stage progressive hormone regulation system and antioxidant protection system of the present invention were used, but a fixed concentration of sucrose (30 parts by weight) was used in each stage, osmotic pressure gradient regulation was not carried out, and maltose was not added to verify the necessity of the osmotic pressure gradient regulation strategy. Other culture conditions were the same as those in Example 3.

[0142] Comparative Example 5: Conventional MS medium and trace element enhancement

[0143] In this comparative example, the three-stage progressive hormone regulation system, antioxidant protection system, and osmotic pressure gradient regulation strategy of the present invention were used, but a conventional MS medium rather than the modified MS medium of the present invention was used, and trace element enhancers (sodium selenite, nickel chloride hexahydrate, and sodium silicate nonahydrate) were not added to verify the role of trace element enhancement. Other culture conditions were the same as those in Example 3.

[0144] A comprehensive comparative evaluation was conducted on Example 3 of the present invention and five comparative examples, using the following test methods and evaluation indicators:

[0145] Embryogenic callus induction rate (%): Calculate the percentage of the number of explants forming embryogenic callus in the total number of inoculated explants. Each treatment was repeated 4 times, with 25 explants inoculated each time.

[0146] Callus formation time (days): Record the time required for 50% of the explants to form obvious callus from the time of inoculation.

[0147] Callus quality score: Score based on the color, compactness and degree of waterlogging of the callus, from 1 to 5 points, with a higher score indicating better quality (5 points: light yellow, highly dense, not waterlogged; 4 points: milky white, dense, slightly waterlogged; 3 points: white, relatively dense, moderately waterlogged; 2 points: transparent, loose, severely waterlogged; 1 point: brown, extremely loose, completely waterlogged).

[0148] Embryogenic potential (%): Transfer the obtained callus to the plant regeneration medium, and calculate the percentage of callus blocks forming somatic embryos after 30 days of culture.

[0149] Genetic stability evaluation: After subculturing the callus 6 times, evaluate its ability to maintain embryogenicity, and calculate the percentage of callus blocks maintaining embryogenicity.

[0150] Peroxide determination: The content of malondialdehyde (MDA) in the callus was determined by the thiobarbituric acid (TBA) method, with the unit of nmol / g fresh weight. The lower the MDA content, the smaller the oxidative damage.

[0151] Through the comprehensive comparison of Example 3 of the present invention and five comparative examples, the experimental results are shown in Table 1:

[0152] Table 1 Comparison between the method of the present invention and the comparative examples in the induction of embryogenic callus of Coffea canephora Pierre var. robusta

[0153]

[0154]

[0155] It can be seen from the experimental results that:

[0156] 1. Compared with the traditional single - hormone system (Comparative Example 1): The three - stage progressive hormone regulation system of the present invention significantly increases the embryogenic callus induction rate (95.0% vs 42.3%), shortens the callus formation time, and greatly improves the callus quality, embryogenic potential and genetic stability. This indicates that precise regulation of hormone levels at different stages is crucial for embryogenic callus induction in Coffea canephora.

[0157] 2. Compared with the simplified two - stage system (Comparative Example 2): The three - stage progressive scheme of the present invention has obvious advantages (induction rate 95.0% vs 63.5%), proving the necessity of the embryogenic induction stage. In the two - stage system, due to the lack of the cytokinin / auxin balance and osmotic pressure environment unique to the embryogenic induction stage, callus directly transitions from dedifferentiation to embryogenic maintenance, resulting in a significant reduction in embryogenic potential and stability.

[0158] 3. Impact of the lack of antioxidant protection system (Comparative Example 3): The absence of antioxidants leads to a significant increase in the MDA content of callus (58.3 vs 18.3 nmol / g), indicating that cells have suffered severe oxidative damage, which directly affects the callus quality (score 2.6 vs 4.8) and embryogenic potential (42.3% vs 86.4%). This result proves that the antioxidant protection system plays a key role in reducing oxidative stress and improving callus quality.

[0159] 4. Importance of osmotic pressure gradient regulation (Comparative Example 4): Compared with a fixed sucrose concentration, osmotic pressure gradient regulation significantly increases the embryogenic callus induction rate (95.0% vs 74.6%) and embryogenic potential (86.4% vs 56.8%). This indicates that timely adjustment of the osmotic pressure environment of the culture medium can effectively promote cell fate transition and embryogenic development.

[0160] 5. Role of trace element enhancement (Comparative Example 5): Although the addition of trace element enhancers has little effect on the induction rate (95.0% vs 85.3%), it significantly improves the callus quality (4.8 vs 4.1 points) and subculture stability (83.5% vs 62.8%). This shows that trace elements such as selenium, nickel and silicon play an important role in maintaining cell viability and genetic stability.

[0161] The above - mentioned comparative experimental results fully verify the scientificity and effectiveness of the comprehensive technical solution of the three - stage progressive hormone regulation system of the present invention combined with antioxidant protection and osmotic pressure gradient regulation. Compared with the prior art, the method of the present invention shows significant advantages in aspects such as embryogenic callus induction rate, callus quality, embryogenic potential and genetic stability, providing reliable technical support for somatic embryogenesis and plant regeneration of Coffea canephora.

Claims

1. A method for inducing embryonic callus of medium-grain coffee, characterized in that: The following steps are involved: Select leaves of coffee of medium grain as explants for surface sterilization; The sterilized explants were cultured in three stages: dedifferentiation stage, embryogenic induction stage and embryogenic maintenance stage; The dedifferentiation phase is cultured under the first culture conditions using a first culture medium supplemented with 2,4-dichlorophenoxyacetic acid; The embryonic induction period is carried out using a second culture medium supplemented with 2,4-dichlorophenoxyacetic acid and 6-benzyladenine under a second culture condition; The embryonic maintenance period adopts a third culture medium supplemented with 1-naphthylacetic acid and kinetin, and is cultured under a third culture condition; Obtain compact embryonic callus with low waterlogging degree.

2. The method for inducing embryonic callus of coffee of cane grain according to claim 1, characterized in that: The first culture medium comprises the following components: Basal culture medium; 2,4-dichlorophenoxyacetic acid 0.8-1.2 parts by weight; Sucrose 25-35 parts by weight; L-glutamine 3.5-4.5 parts by weight; 0.8-1.2 parts by weight of activated carbon; Agar 6.5-7.5 parts by weight.

3. The method for inducing embryonic callus of coffee of cane grain variety according to claim 1, characterized in that: The second culture medium comprises the following components: Basal culture medium; 2,4-dichlorophenoxyacetic acid 0.4-0.6 parts by weight; 2.0-2.5 parts by weight of 6-benzyladenine; Sucrose 35-45 parts by weight; 4.5-5.5 parts by weight of hydrolyzed casein; L-proline 4.5-5.5 parts by weight; Ascorbic acid 0.9-1.1 parts by weight; Polyethylene glycol 4000 3.5-4.5 parts by weight; 2.0-3.0 parts by weight of vegetable gel.

4. The method for inducing embryonic callus of coffee of cane grain according to claim 1, characterized in that: The third culture medium comprises the following components: Basal culture medium; 1-naphthylacetic acid 0.2-0.3 parts by weight; Kinetin 0.9-1.1 parts by weight; Sucrose 25-35 parts by weight; 8-12 parts by weight of maltose; Cystine 0.8-1.2 parts by weight; Glutathione 0.08-0.12 parts by weight; Vitamin E 0.008-0.012 parts by weight; 2.0-3.0 parts by weight of vegetable gel.

5. The method for inducing embryonic callus of coffee of any one of claims 1 to 4, characterized in that: The basic culture medium is a modified MS culture medium, comprising the following components: 15-20 parts by weight of ammonium nitrate; Potassium nitrate 18-22 parts by weight; 3.5-4.5 parts by weight of magnesium sulfate heptahydrate; 1.5-2.5 parts by weight of potassium dihydrogen phosphate; Calcium chloride dihydrate 3.0-4.0 parts by weight; MS trace elements; Iron source; Organic components; The MS trace elements include: 1.5-2.2 parts by weight of manganese sulfate monohydrate, 0.8-1.2 parts by weight of zinc sulfate heptahydrate, 0.5-0.7 parts by weight of boric acid, 0.07-0.09 parts by weight of potassium iodide, 0.02-0.03 parts by weight of sodium molybdate dihydrate, 0.002-0.003 parts by weight of copper sulfate pentahydrate, and 0.002-0.003 parts by weight of cobalt chloride hexahydrate; the iron source includes: 3.5-4.5 parts by weight of EDTA-Na2, 2.5-3.5 parts by weight of ferrous sulfate heptahydrate; the organic components include: 9-11 parts by weight of inositol, 0.09-0.11 parts by weight of thiamine hydrochloride, 0.045-0.055 parts by weight of pyridoxine hydrochloride, 0.045-0.055 parts by weight of nicotinic acid, and 0.18-0.22 parts by weight of glycine.

6. The method for inducing embryonic callus of coffee of cane grain according to claim 5, characterized in that: The basic medium also includes the following trace element enhancers: Sodium selenate 0.0008-0.0012 parts by weight; Nickel chloride hexahydrate 0.0003-0.0007 parts by weight; Sodium silicate nonahydrate 0.08-0.12 parts by weight.

7. The method for inducing embryonic callus of coffee of cane grain according to claim 1, characterized in that: The first culture condition is: temperature 25±1°C, complete darkness culture for 7-10 days, then switching to a 16 / 8 hour light / dark cycle, light intensity 20-30 μmol·m-2·s-1, pH 5.6-5.8, and culture for 21-28 days.

8. The method for inducing embryonic callus of coffee of cane grain according to claim 1, characterized in that: The second culture conditions are: temperature 26±1°C, 12 / 12 hour light / dark cycle, light intensity 40-50 μmol·m-2·s-1, pH 5.4-5.6, and culture for 14-21 days.

9. The method for inducing embryonic callus of coffee of cane grain according to claim 1, characterized in that: The third culture condition is: temperature 24±1°C, 16 / 8 hour light / dark cycle, light intensity 50-60 μmol·m-2·s-1, pH 5.2-5.4, and culture for 21-28 days.

10. The method for inducing embryonic callus of coffee of cane grain according to claim 1, characterized in that: The surface sterilization steps include: soaking in 75% ethanol for 30-40 seconds; soaking in 0.1% mercuric chloride solution for 7-9 minutes; treating with 0.5-1.0% sodium hypochlorite solution for 10-12 minutes; washing with sterile water for 5-6 times, each time for 3-5 minutes.

Images