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] This invention relates to the field of plant tissue culture technology, specifically to a method for inducing embryogenic callus in medium-sized coffee seeds. Background Technology

[0002] Robusta coffee (C. canephora Pierre ex A. Froehner) is one of the world's most important economic crops, accounting for over 45% of the global coffee industry's cultivated area and yield. Currently, the propagation of Robusta coffee mainly relies on grafting and cutting propagation. These traditional propagation methods have disadvantages such as low propagation coefficient and long growth cycle, making it difficult to meet the large-scale demand for high-quality seedlings in the modern coffee industry.

[0003] Somatic embryogenesis, as a highly efficient asexual reproduction technique, holds significant promise for the rapid propagation and germplasm preservation of medium-sized coffee varieties. The induction of embryogenic callus is a crucial step in somatic embryogenesis, directly impacting the quantity and quality of subsequent plant regeneration. However, current techniques for inducing embryogenic callus in medium-sized coffee varieties suffer from low induction rates, poor callus quality, and insufficient genetic stability.

[0004] The reported techniques for inducing embryogenic callus in medium-sized coffee varieties have the following shortcomings: First, most methods employ single or dual hormone regulation systems, lacking precise control over different developmental stages; second, insufficient optimization of culture environment parameters leads to severe waterlogging of callus tissue; and third, a lack of systematic solutions targeting oxidative stress and osmotic pressure regulation results in unstable callus quality. Therefore, there is an urgent need to develop an efficient and stable method for inducing embryogenic callus in medium-sized coffee varieties. Summary of the Invention

[0005] In view of the shortcomings of the prior art, the purpose of this invention is to provide a method for efficiently inducing embryogenic callus in medium-grain coffee. This method significantly improves the induction efficiency and quality of embryogenic callus by using a three-stage progressive hormone regulation system combined with antioxidant protection and osmotic pressure gradient regulation strategy.

[0006] The purpose of this invention is to provide a method for inducing embryogenic callus in medium-sized coffee seeds, comprising the following steps: Medium-sized coffee leaves were selected as explants for surface sterilization. The sterilized explants were cultured in three stages: dedifferentiation, embryogenicity induction, and embryogenicity maintenance. The dedifferentiation period was carried out using a first culture medium supplemented with 2,4-dichlorophenoxyacetic acid, and cultured under the first culture conditions. The embryonic induction period was carried out using a second culture medium supplemented with 2,4-dichlorophenoxyacetic acid and 6-benzyladenine, and cultured under the second culture conditions. The embryogenicity maintenance period was carried out using a third culture medium supplemented with 1-naphthacetic acid and kinetin, and cultured under third culture conditions; Obtain embryogenic callus with firm texture and low degree of water immersion.

[0007] Specifically, the first culture medium comprises the following components: Basic culture medium; 0.8-1.2 parts by weight of 2,4-dichlorophenoxyacetic acid; 25-35 parts by weight of sucrose; L-glutamine 3.5-4.5 parts by weight; Activated carbon 0.8-1.2 parts by weight; 6.5-7.5 parts by weight of agar.

[0008] Specifically, the second culture medium comprises the following components: Basic culture medium; 0.4-0.6 parts by weight of 2,4-dichlorophenoxyacetic acid; 6-Benzyladenine 2.0-2.5 parts by weight; 35-45 parts by weight of sucrose; Hydrolyzed casein 4.5-5.5 parts by weight; 4.5-5.5 parts by weight of L-proline; Ascorbic acid 0.9-1.1 parts by weight; Polyethylene glycol 4000: 3.5-4.5 parts by weight; Plant-based gel, 2.0-3.0 parts by weight.

[0009] Specifically, the third culture medium comprises the following components: Basic culture medium; 0.2-0.3 parts by weight of 1-naphthaleneacetic acid; Kinetin 0.9-1.1 parts by weight; 25-35 parts by weight of sucrose; 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; Plant-based gel, 2.0-3.0 parts by weight.

[0010] Specifically, the basal culture medium is a modified MS medium, comprising the following components: 15-20 parts by weight of ammonium nitrate; 18-22 parts by weight of potassium nitrate; Magnesium sulfate heptahydrate 3.5-4.5 parts by weight; 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, 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.

[0011] Specifically, the basal culture 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.

[0012] Specifically, the first culture conditions are: temperature 25±1℃, cultured in complete darkness for 7-10 days, then switched to a 16 / 8 hour light / dark cycle, with light intensity... The pH value is 5.6-5.8, and the culture time is 21-28 days.

[0013] Specifically, the second culture conditions are: temperature 26±1℃, 12 / 12 hour light / dark cycle, and light intensity... The pH value is 5.4-5.6, and the culture time is 14-21 days.

[0014] Specifically, the third culture condition is: temperature 24±1℃, 16 / 8 hour light / dark cycle, light intensity... The pH value is 5.2-5.4, and the culture time is 21-28 days.

[0015] Specifically, the surface sterilization steps include: immersing in 75% ethanol for 30-40 seconds; immersing in 0.1% mercuric chloride solution for 7-9 minutes; treating with 0.5-1.0% sodium hypochlorite solution for 10-12 minutes; and washing with sterile water 5-6 times, 3-5 minutes each time.

[0016] Compared with the prior art, the present invention has the following beneficial effects: 1. Through a three-stage progressive hormone regulation, the induction rate of embryogenic callus tissue reached 85-95%, significantly higher than the 30-50% of existing technologies; 2. By employing a synergistic mechanism of antioxidant protection and osmotic pressure gradient regulation, the induced embryogenic callus exhibits excellent characteristics such as firmness, low degree of water immersion, and high embryogenic potential. 3. Through nitrogen source balance and synergistic effect of trace elements, the cultured embryogenic callus can be stably subcultured 6-8 times without losing embryogenicity, providing high-quality starting material for large-scale asexual propagation of medium-grain coffee. 4. The optimized physical environment factor precise control scheme improves the controllability and repeatability of the cultivation process, laying the foundation for the large-scale production of callus tissue from medium-sized coffee embryos. Detailed Implementation

[0017] The present invention will be further described in detail below with reference to specific embodiments, but the scope of protection of the present invention is not limited thereto.

[0018] This invention provides a method for inducing embryogenic callus in medium-sized coffee varieties, comprising the following steps: selecting medium-sized coffee leaves as explants and performing surface sterilization; sequentially culturing the sterilized explants through three stages: dedifferentiation stage, embryogenic induction stage, and embryogenic maintenance stage; the dedifferentiation stage is culturing in a first culture medium supplemented with 2,4-dichlorophenoxyacetic acid under first culture conditions; the embryogenic induction stage is culturing in a second culture medium supplemented with 2,4-dichlorophenoxyacetic acid and 6-benzyladenine under second culture conditions; the embryogenic maintenance stage is culturing in a third culture medium supplemented with 1-naphthaleneacetic acid and kinetin under third culture conditions; and obtaining compact embryogenic callus tissue with low waterlogging.

[0019] This invention employs a three-stage progressive hormone regulation system, scientifically dividing the induction process of embryogenic callus into three phases: dedifferentiation, embryogenic induction, and embryogenic maintenance. Specific culture medium formulations and conditions are designed for each phase. Specifically, the dedifferentiation phase primarily aims to promote cell dedifferentiation with high concentrations of 2,4-D; the embryogenic induction phase induces embryogenic potential through an optimized cytokinin / auxin ratio (approximately 4:1); and the embryogenic maintenance phase uses a combination of NAA and KT to maintain the stability of the embryogenic state. The orderly transition between these three phases ensures an efficient transformation from ordinary somatic cells to embryogenic cells.

[0020] Meanwhile, the synergistic mechanism of antioxidant protection and osmotic pressure gradient regulation in this invention solves the problems of severe callus hydration and embryogenic instability in traditional methods. By adding specific antioxidants (such as activated charcoal, ascorbic acid, glutathione, and vitamin E) at different stages, a multi-layered antioxidant defense system is constructed; combined with a carbon source gradient regulation strategy (staged adjustment of sucrose concentration and introduction of maltose), an osmotic environment conducive to embryogenic development is created.

[0021] To further optimize the cultivation effect, this invention also employs a nitrogen source balance and trace element synergistic enhancement strategy. By rationally combining organic nitrogen sources such as L-glutamine, hydrolyzed casein, L-proline, and cysteine, nitrogen utilization efficiency is improved; simultaneously, trace element enhancers such as selenium, nickel, and silicon are introduced to promote the activation of specific metabolic pathways.

[0022] The compounds used in this invention are summarized below: ammonium nitrate potassium nitrate Magnesium sulfate heptahydrate Potassium dihydrogen phosphate Calcium chloride dihydrate Manganese sulfate monohydrate Zinc sulfate heptahydrate Boric acid Potassium iodide Sodium molybdate dihydrate Copper sulfate pentahydrate Cobalt chloride hexahydrate , Ferrous sulfate heptahydrate Inositol Thiamine hydrochloride (vitamin) ), pyridoxine hydrochloride (vitamin) ), niacin, glycine 2,4-Dichlorophenoxyacetic acid ,sucrose L-glutamine Activated carbon (Sigma-Aldrich, C9157), agar (Sigma-Aldrich, A1296), 6-benzyladenine (BA or 6-BA), hydrolyzed casein (Sigma-Aldrich, C7290), L-proline Ascorbic acid (vitamin C), polyethylene glycol 4000 (PEG 4000), plant gel (Phytagel, Sigma-Aldrich, P8169), 1-naphthaleneacetic acid (NAA), kinetin (KT or Kinetin), maltose Cystine Glutathione Vitamin E (tocopherol acetate), sodium selenite Nickel chloride hexahydrate Sodium silicate nonahydrate Mercuric chloride Sodium hypochlorite (NaClO), ethanol, thiobarbituric acid (TBA).

[0023] Example 1: A method for inducing embryogenic callus in medium-sized coffee varieties, comprising the following steps: (1) Selection and sterilization of explants: Healthy plants of the medium-sized coffee variety (C. canephora Pierre ex A. Froehner) that have been growing for 3 months were selected. The second pair of fully expanded leaves were taken and cut into 1.5 cm² square segments. The following sterilization procedure was used: soaking in 75% ethanol for 30 seconds, soaking in 0.1% mercuric chloride solution for 7 minutes, treating with 0.5% sodium hypochlorite solution for 10 minutes, and washing with sterile water 5 times, 3 minutes each time.

[0024] (2) Dedifferentiation stage culture: Sterilized explants were inoculated with the cut edges facing down onto the first culture medium. The first culture medium included the following components: basal culture medium; 0.8 parts by weight of 2,4-dichlorophenoxyacetic acid; 25 parts by weight of sucrose; 3.5 parts by weight of L-glutamine; 0.8 parts by weight of activated carbon; and 6.5 parts by weight of agar. Among them, the basal culture medium was a modified MS medium, including the following components: 15 parts by weight of ammonium nitrate; 18 parts by weight of potassium nitrate; 3.5 parts by weight of magnesium sulfate heptahydrate; 1.5 parts by weight of potassium dihydrogen phosphate; 3.0 parts by weight of calcium chloride dihydrate; MS trace elements; iron source; organic components; MS trace elements included: 1.5 parts by weight of manganese sulfate monohydrate, 0.8 parts by weight of zinc sulfate heptahydrate, 0.5 parts by weight of boric acid, 0.07 parts by weight of potassium iodide, 0.02 parts by weight of sodium molybdate dihydrate, 0.002 parts by weight of copper sulfate pentahydrate, and 0.002 parts by weight of cobalt chloride hexahydrate; Iron sources include: 3.5 parts by weight, 2.5 parts by weight of ferrous sulfate heptahydrate; 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.

[0025] The initial culture conditions were: temperature 25℃, complete darkness for 7 days, followed by a 16 / 8 hour light / dark cycle, with a light intensity of [missing information]. pH 5.6, cultured for 21 days.

[0026] During this stage, 2,4-dichlorophenoxyacetic acid, as a potent auxin, can disrupt cell differentiation, promoting dedifferentiation and division. A low concentration of sucrose (25 parts by weight) provides a suitable carbon source and osmotic pressure environment for the initial stage of cell division. L-glutamine, as an readily available nitrogen source, accelerates cell metabolism and protein synthesis. The addition of activated charcoal adsorbs harmful phenolic substances and peroxides generated during culture, mitigating oxidative stress. After 21 days of culture, dense, milky-white callus tissue can be observed at the explant incision site.

[0027] (3) Embryogenic induction culture: The formed callus tissue was transferred to the second culture medium. The second culture medium consisted of 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; and 2.0 parts by weight of plant gel. The second culture conditions were: temperature 26℃, 12 / 12-hour light / dark cycle, and light intensity... pH 5.4, cultured for 14 days.

[0028] During this stage, the concentration of 2,4-dichlorophenoxyacetic acid decreased (0.4 parts by weight) while the concentration of 6-benzyladenine increased (2.0 parts by weight), resulting in a cytokinin / auxin ratio of approximately 4:1, a ratio proven to favor the expression of genes related to somatic embryogenesis. Increased sucrose concentration (35 parts by weight) created a mild osmotic stress environment, promoting embryogenic differentiation. The addition of hydrolyzed casein and L-proline provided a diverse amino acid profile, with L-proline also acting as an osmotic regulator. Ascorbic acid, as a direct free radical scavenger, protected cells from oxidative damage. Polyethylene glycol 4000 maintained osmotic pressure while promoting intercellular signaling. After 14 days of culture, a yellowish-white granular structure was observed on the callus surface, a preliminary characteristic of embryogenic development.

[0029] (4) Embryogenic maintenance culture: Callus containing granular structures was transferred to the third culture medium. The third culture medium consisted of the following components: basal medium; 0.2 parts by weight of 1-naphthaleneacetic acid; 0.9 parts by weight of kinetin; 25 parts by weight of sucrose; 8 parts by weight of maltose; 0.8 parts by weight of cystine; 0.08 parts by weight of glutathione; 0.008 parts by weight of vitamin E; and 2.0 parts by weight of plant gel. The third culture conditions were: temperature 24℃, 16 / 8 hour light / dark cycle, and light intensity... pH 5.2, cultured for 21 days.

[0030] During the embryogenic maintenance phase, the combination of 1-naphthaleneacetic acid (NAA) and kinetin stabilizes the embryogenic state and prevents early differentiation. A 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 glutathione and vitamin E, as potent antioxidants, form a complete antioxidant defense system. After 21 days of culture, high-quality embryogenic callus is obtained, exhibiting a milky white to pale yellow color, high density, smooth surface, low degree of hydration, and distinct embryogenic characteristics.

[0031] In this embodiment, the induction rate of embryogenic callus reached 86%, the callus quality was excellent, and the subsequent somatic cell embryo differentiation rate reached 74%, which is much higher than the 30-40% of the traditional method.

[0032] Example 2: A method for inducing embryogenic callus in medium-sized coffee varieties, comprising the following steps: (1) Selection and sterilization of explants: Healthy plants of the medium-sized coffee variety (C. canephora Pierre ex A. Froehner) that have been growing for 4 months were selected. The third pair of fully expanded leaves were taken and cut into 2.0 cm² square segments. The following sterilization procedure was used: soaking in 75% ethanol for 40 seconds, soaking in 0.1% mercuric chloride solution for 9 minutes, treating with 1.0% sodium hypochlorite solution for 12 minutes, and washing with sterile water 6 times, 5 minutes each time.

[0033] (2) Dedifferentiation stage culture: Sterilized explants were inoculated onto the first culture medium. The first culture medium consisted of the following components: basal medium; 1.2 parts by weight of 2,4-dichlorophenoxyacetic acid; 35 parts by weight of sucrose; 4.5 parts by weight of L-glutamine; 1.2 parts by weight of activated carbon; and 7.5 parts by weight of agar. Among them, the basal medium consisted of the following components: 20 parts by weight of ammonium nitrate; 22 parts by weight of potassium nitrate; 4.5 parts by weight of magnesium sulfate heptahydrate; 2.5 parts by weight of potassium dihydrogen phosphate; 4.0 parts by weight of calcium chloride dihydrate; MS trace elements; iron source; and organic components. MS trace elements include: 2.2 parts by weight of manganese sulfate monohydrate, 1.2 parts by weight of zinc sulfate heptahydrate, 0.7 parts by weight of boric acid, 0.09 parts by weight of potassium iodide, 0.03 parts by weight of sodium molybdate dihydrate, 0.003 parts by weight of copper sulfate pentahydrate, and 0.003 parts by weight of cobalt chloride hexahydrate. Iron sources include: 4.5 parts by weight, 3.5 parts by weight of ferrous sulfate heptahydrate; The organic components include: 11 parts by weight of inositol, 0.11 parts by weight of thiamine hydrochloride, 0.055 parts by weight of pyridoxine hydrochloride, 0.055 parts by weight of nicotinic acid, and 0.22 parts by weight of glycine. Including 0.0012 parts by weight of sodium selenate; 0.0007 parts by weight of nickel chloride hexahydrate; and 0.12 parts by weight of sodium silicate nonahydrate. The initial culture conditions were: temperature 25℃, complete darkness for 10 days, followed by a 16 / 8 hour light / dark cycle, with light intensity... pH 5.8, cultured for 28 days.

[0034] (3) Embryogenic induction culture: The formed callus tissue was transferred to the second culture medium. The second culture medium consisted of the following components: basal medium; 0.6 parts 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; and 3.0 parts by weight of plant gel. The second culture conditions were: temperature 26℃, 12 / 12-hour light / dark cycle, and light intensity... pH 5.6, cultured for 21 days.

[0035] (4) Embryogenic maintenance culture: Callus containing granular structures was transferred to the third culture medium. The third culture medium consisted of the following components: basal medium; 0.3 parts by weight of 1-naphthaleneacetic acid; 1.1 parts 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 parts by weight of glutathione; 0.012 parts by weight of vitamin E; and 3.0 parts by weight of plant gel. The third culture conditions were: temperature 24℃, 16 / 8 hour light / dark cycle, and light intensity... pH 5.4, cultured for 28 days.

[0036] In this embodiment, the upper limits of each component were used, and a trace element enhancer was added. The dedifferentiation effect was enhanced by increasing the concentration of 2,4-D (1.2 parts by weight) and extending the dark culture time (10 days); the embryogenic induction effect was enhanced by increasing the concentration of cytokinin (2.5 parts by weight of 6-BA) and sucrose (45 parts by weight); and the cell protection effect during the embryogenic maintenance stage was enhanced by increasing the concentration of antioxidants (0.12 parts by weight of glutathione and 0.012 parts by weight of vitamin E). This embodiment is particularly suitable for coffee varieties with poor induction response, achieving an embryogenic callus induction rate of 90%, and the callus can be stably subcultured more than 8 times.

[0037] Example 3: A method for inducing embryogenic callus in medium-sized coffee varieties, comprising the following steps: (1) Selection and sterilization of explants: Healthy plants of the medium-sized coffee variety (C. canephora Pierre ex A. Froehner) that have been growing for 3.5 months were selected. The 2nd to 3rd pairs of fully expanded leaves were taken and cut into 1.8 cm² square segments. The following sterilization procedure was used: soaking in 75% ethanol for 35 seconds, soaking in 0.1% mercuric chloride solution for 8 minutes, treating with 0.8% sodium hypochlorite solution for 11 minutes, and washing with sterile water 5 times, 4 minutes each time.

[0038] (2) Dedifferentiation stage culture: Sterilized explants were inoculated onto the first culture medium. The first culture medium consisted of the following components: basal culture medium; 1.0 part by weight of 2,4-dichlorophenoxyacetic acid; 30 parts by weight of sucrose; 4.0 parts by weight of L-glutamine; 1.0 part by weight of activated carbon; and 7.0 parts by weight of agar. Among them, the basal culture medium consisted of the following components: 16.5 parts by weight of ammonium nitrate; 19 parts by weight of potassium nitrate; 3.7 parts by weight of magnesium sulfate heptahydrate; 1.7 parts by weight of potassium dihydrogen phosphate; 3.3 parts by weight of calcium chloride dihydrate; MS trace elements; iron source; organic components; and 0.001 parts by weight of sodium selenate; 0.0005 parts by weight of nickel chloride hexahydrate; and 0.1 parts by weight of sodium silicate nonahydrate.

[0039] MS trace elements include: 1.85 parts by weight of manganese sulfate monohydrate, 1.0 part by weight of zinc sulfate heptahydrate, 0.6 parts by weight of boric acid, 0.08 parts by weight of potassium iodide, 0.025 parts by weight of sodium molybdate dihydrate, 0.0025 parts by weight of copper sulfate pentahydrate, and 0.0025 parts by weight of cobalt chloride hexahydrate. Iron sources include: 4.0 parts by weight, 3.0 parts by weight of ferrous sulfate heptahydrate; The organic components include: 10 parts by weight of inositol, 0.1 parts by weight of thiamine hydrochloride, 0.05 parts by weight of pyridoxine hydrochloride, 0.05 parts by weight of nicotinic acid, and 0.2 parts by weight of glycine; The initial culture conditions were: temperature 25℃, complete darkness for 8 days, followed by a 16 / 8 hour light / dark cycle, with a light intensity of [missing information]. pH 5.7, cultured for 24 days.

[0040] (3) Embryogenic induction culture: The formed callus tissue was transferred to the second culture medium. The second culture medium consisted of the following components: basal medium; 0.5 parts 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; and 2.5 parts by weight of plant gel. The second culture conditions were: temperature 26℃, 12 / 12-hour light / dark cycle, and light intensity... pH 5.5, cultured for 18 days.

[0041] (4) Embryogenic maintenance culture: Callus containing granular structures was transferred to the third culture medium. The third culture medium consisted of the following components: basal medium; 0.25 parts by weight of 1-naphthaleneacetic 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 parts by weight of vitamin E; and 2.5 parts by weight of plant gel. The third culture conditions were: temperature 24℃, 16 / 8 hour light / dark cycle, and light intensity... pH 5.3, cultured for 25 days.

[0042] This embodiment uses the median 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) to sucrose (30 parts by weight) creates a suitable dedifferentiation environment; the approximately 4:1 ratio of 2,4-D (0.5 parts by weight) to 6-BA (2.2 parts by weight) optimizes the embryogenicity induction effect; the combination of NAA (0.25 parts by weight) and KT (1.0 part by weight), and the synergistic effect of glutathione (0.1 parts by weight) and vitamin E (0.01 parts by weight), form a complete embryogenicity maintenance and protection system. Under this scheme, the embryogenic callus induction rate reaches 95%, the embryogenicity quality is optimal, exhibiting highly dense pale yellow tissue that is completely non-waterlogged, and the highest embryogenicity stability.

[0043] Example 4: A method for inducing embryogenic callus in medium-sized coffee varieties, comprising the following steps: (1) Selection and sterilization of explants: Healthy plants of the medium-sized coffee variety (C. canephora Pierre ex A. Froehner) that have been growing for 3.5 months were selected. The third pair of fully expanded leaves were taken and cut into 1.7 cm² square segments. The following sterilization procedure was used: soaking in 75% ethanol for 35 seconds, soaking in 0.1% mercuric chloride solution for 8 minutes, treating with 0.7% sodium hypochlorite solution for 11 minutes, and washing with sterile water 5 times, 4 minutes each time.

[0044] (2) Dedifferentiation stage culture: Sterilized explants were inoculated onto the first culture medium. The first culture medium consisted of the following components: basal culture 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; and 7.0 parts by weight of agar. Among them, the basal culture medium consisted of 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 parts by weight of sodium selenate; 0.0005 parts by weight of nickel chloride hexahydrate; and 0.1 parts by weight of sodium silicate nonahydrate.

[0045] 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, and 0.0022 parts by weight of cobalt chloride hexahydrate. Iron sources include: 3.7 parts by weight, 2.7 parts by weight of ferrous sulfate heptahydrate; 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, and 0.19 parts by weight of glycine; The initial culture conditions were: temperature 24℃, complete darkness for 9 days, followed by a 16 / 8 hour light / dark cycle, with a light intensity of [missing information]. pH 5.7, cultured for 25 days.

[0046] In this embodiment, the temperature (24°C) and sucrose concentration (25 parts by weight) during the dedifferentiation period were specifically reduced, considering that the (C. canephora Pierre ex A. Froehner) variety has a better dedifferentiation effect at lower temperatures.

[0047] (3) Embryogenic induction culture: The formed callus tissue was transferred to the second culture medium. The second culture medium consisted of the following components: basal medium; 0.4 parts 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; and 2.5 parts by weight of plant gel. The second culture conditions were: temperature 27℃, 12 / 12-hour light / dark cycle, and light intensity... pH 5.5, cultured for 18 days.

[0048] In this embodiment, the temperature during the embryonic induction period was specifically increased (27°C) because the variety (C. canephora Pierre ex A. Froehner) requires a higher temperature to activate relevant metabolic pathways during the embryonic induction stage.

[0049] (4) Embryogenic maintenance culture: Callus containing granular structures was transferred to the third culture medium. The third culture medium consisted of the following components: basal medium; 0.25 parts by weight of 1-naphthaleneacetic 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 parts by weight of vitamin E; and 2.5 parts by weight of plant gel. The third culture conditions were: temperature 24℃, 16 / 8 hour light / dark cycle, and light intensity... pH 5.3, cultured for 25 days.

[0050] This embodiment specifically optimized the temperature parameters for the physiological characteristics of the C. canephora Pierre ex A. Froehner variety, achieving an embryogenic callus induction rate of 88% and good callus quality, making it suitable for constructing a somatic embryogenesis system for this variety.

[0051] Example 5: A method for inducing embryogenic callus in medium-sized coffee varieties, comprising the following steps: (1) Selection and sterilization of explants: Healthy plants of the medium-sized coffee variety (C. canephora Pierre ex A. Froehner) that have been growing for 4 months were selected. The second pair of fully expanded leaves were taken and cut into 1.6 cm² square segments. The following sterilization procedure was used: soaking in 75% ethanol for 32 seconds, soaking in 0.1% mercuric chloride solution for 8 minutes, treating with 0.6% sodium hypochlorite solution for 10 minutes, and washing with sterile water 5 times, 3 minutes each time.

[0052] (2) Dedifferentiation stage culture: Sterilized explants were inoculated onto the first culture medium. The first culture medium consisted of the following components: 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; and 7.0 parts by weight of agar. Among them, the basal medium consisted of 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; iron source; organic components; and 0.001 parts by weight of sodium selenate; 0.0005 parts by weight of nickel chloride hexahydrate; and 0.1 parts by weight of sodium silicate nonahydrate.

[0053] MS trace elements include: 2.0 parts by weight of manganese sulfate monohydrate, 1.1 parts by weight of zinc sulfate heptahydrate, 0.65 parts by weight of boric acid, 0.085 parts by weight of potassium iodide, 0.028 parts by weight of sodium molybdate dihydrate, 0.0028 parts by weight of copper sulfate pentahydrate, and 0.0028 parts by weight of cobalt chloride hexahydrate. Iron sources include: 4.3 parts by weight, 3.3 parts by weight of ferrous sulfate heptahydrate; The organic components include: 10.5 parts by weight of inositol, 0.105 parts by weight of thiamine hydrochloride, 0.052 parts by weight of pyridoxine hydrochloride, 0.052 parts by weight of nicotinic acid, and 0.21 parts by weight of glycine. The first culture conditions were: temperature 25℃, cultured in complete darkness for 8 days, then switched to a 16 / 8 hour light / dark cycle, with light intensity... pH 5.7, cultured for 24 days.

[0054] (3) Embryogenic induction culture: The formed callus tissue was transferred to the second culture medium. The second culture medium consisted of the following components: basal medium; 0.45 parts 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; and 2.4 parts by weight of plant gel. The second culture conditions were: temperature 26℃, 12 / 12-hour light / dark cycle, and light intensity... pH 5.5, cultured for 16 days.

[0055] (4) Embryogenic maintenance culture: Callus containing granular structures was transferred to the third culture medium. The third culture medium consisted of the following components: 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; and 2.3 parts by weight of plant gel. The third culture conditions were: temperature 24℃, 16 / 8 hour light / dark cycle, and light intensity... pH 5.3, cultured for 22 days.

[0056] This embodiment addresses the resistance characteristics of the medium-sized coffee variety by appropriately reducing the concentration of some components. As a rust-resistant variety, medium-sized coffee has thicker cell walls and relatively lower embryogenic development potential. By fine-tuning the hormone concentration and culture time, the induction rate of embryogenic callus was successfully increased to 82%, providing a reliable method for somatic embryogenesis in this disease-resistant variety.

[0057] The above embodiments demonstrate that the method for inducing embryogenic callus in medium-sized coffee varieties provided by this invention is highly efficient, stable, and adaptable. It can be appropriately adjusted according to the physiological characteristics of different varieties to achieve efficient induction of high-quality embryogenic callus. This invention solves the problems of low induction rate, poor callus quality, and insufficient genetic stability in existing technologies for medium-sized coffee varieties, providing key technical support for somatic embryogenesis and large-scale asexual reproduction in medium-sized coffee varieties.

[0058] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. The scope of protection of the present invention is defined by the claims.

[0059] To verify the superiority of the three-stage progressive hormone regulation system proposed in this invention, which combines antioxidant protection with osmotic pressure gradient regulation strategy, the following comparative experiment was designed.

[0060] Comparative Example 1: Traditional Single Hormone System This comparative study employed a traditional single-hormone system for inducing callus formation in medium-sized coffee germ. The specific method is as follows: Leaves of the medium-sized coffee variety (C. canephora Pierre ex A. Froehner) were selected as explants, treated with standard sterilization procedures, and then inoculated onto MS medium containing 1.0 part by weight of 2,4-D and 0.5 parts by weight of BAP. The medium also contained 30 parts by weight of sucrose and 7.0 parts by weight of agar, and the pH was adjusted to 5.7. The culture conditions were: temperature 25℃, photoperiod 16 / 8 hours (light / dark), and light intensity... After 60 days of culture, the formation of callus tissue was observed.

[0061] Comparative Example 2: Two-stage culture system This comparative example uses a simplified two-stage culture system for inducing embryogenic callus in medium-sized coffee seeds. The first stage uses a high concentration of 2,4-D for dedifferentiation induction, and the second stage directly uses cytokinin to maintain embryogenicity, omitting the embryogenic induction period described in this invention. The specific method is as follows: Dedifferentiation stage: Sterilized leaves of the medium-sized coffee variety (C. canephora Pierre ex A. Froehner) were inoculated onto 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, at pH 5.7. The culture conditions were 25°C, followed by 8 days of complete darkness, then a 16 / 8 hour (light / dark) photoperiod, with a light intensity of... , Cultivate for 30 days.

[0062] Embryogenic maintenance phase: The formed callus tissue was transferred to 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 plant gel, at pH 5.3. Culture conditions were: temperature 24℃, 16 / 8 hour (light / dark) photoperiod, and light intensity... , Cultivate for 30 days.

[0063] Comparative Example 3: Lack of antioxidant protection system This comparative example uses the three-stage progressive hormone regulation system of the present invention, but without the addition of antioxidants (including activated charcoal, ascorbic acid, glutathione, and vitamin E) at each stage, to verify the importance of the antioxidant protection system. Other culture conditions are the same as in Example 3.

[0064] Comparative Example 4: Lack of osmotic pressure gradient regulation This comparative example employs the three-stage progressive hormone regulation system and antioxidant protection system of the present invention, but uses a fixed concentration of sucrose (30 parts by weight) in each stage, without osmotic gradient regulation or the addition of maltose, to verify the necessity of the osmotic gradient regulation strategy. Other culture conditions are the same as in Example 3.

[0065] Comparative Example 5: Conventional MS medium with micronutrient enhancement This comparative example employs the three-stage progressive hormone regulation system, antioxidant protection system, and osmotic pressure gradient regulation strategy of this invention, but uses conventional MS medium instead of the modified MS medium of this invention, and does not add trace element enhancers (sodium selenate, nickel chloride hexahydrate, and sodium silicate nonahydrate) to verify the effect of trace element enhancement. Other culture conditions are the same as in Example 3.

[0066] The present invention's Embodiment 3 was comprehensively compared and evaluated with five comparative examples, using the following test methods and evaluation indicators: Embryogenic callus induction rate (%): Calculate the percentage of explants that form embryogenic callus out of the total number of explants inoculated. Each treatment was repeated 4 times, with 25 explants inoculated each time.

[0067] Callus formation time (days): Record the time required from inoculation to the formation of 50% of the explants as visible callus.

[0068] Callus quality scoring: The callus is scored based on its color, firmness, and degree of hydration, ranging from 1 to 5 points. The higher the score, the better the quality (5 points: pale yellow, highly dense, no hydration; 4 points: milky white, dense, slightly hydrated; 3 points: white, relatively dense, moderately hydrated; 2 points: transparent, loose, severely hydrated; 1 point: brown, extremely loose, completely hydrated).

[0069] Embryogenic potential (%): The percentage of callus tissue that forms somatic embryos is calculated after transferring the obtained callus tissue to plant regeneration medium and culturing for 30 days.

[0070] Genetic stability evaluation: After six subcultures of callus, its ability to maintain embryogenicity was assessed, and the percentage of callus blocks that maintained embryogenicity was calculated.

[0071] Peroxide determination: The malondialdehyde (MDA) content in callus was determined using the thiobarbituric acid (TBA) method, with units of nmol / g fresh weight. The lower the MDA content, the less oxidative damage.

[0072] The experimental results, obtained through a comprehensive comparison of Embodiment 3 of the present invention with five comparative examples, are shown in Table 1: Table 1. Comparison of the method of the present invention and the comparative example in the induction of embryogenic callus in medium-grain coffee.

[0073] The experimental results show that: 1. Compared with traditional single-hormone systems (Comparative Example 1): The three-stage progressive hormone regulation system of this invention significantly improved the embryogenic callus induction rate (95.0% vs 42.3%), shortened the callus formation time, and greatly improved callus quality, embryogenic potential, and genetic stability. This indicates that precise staged regulation of hormone levels is crucial for embryogenic callus induction in medium-grain coffee.

[0074] 2. Compared with the simplified two-stage system (Comparative Example 2): The three-stage progressive scheme of this invention has significant advantages (induction rate 95.0% vs 63.5%), demonstrating the necessity of the embryogenic induction period. In the two-stage system, due to the lack of the cytokinin / auxin balance and osmotic pressure environment unique to the embryogenic induction period, the callus tissue directly transitions from dedifferentiation to embryogenic maintenance, resulting in a significant reduction in embryogenic potential and stability.

[0075] 3. Effects of lack of antioxidant protection system (Comparative Example 3): The absence of antioxidants led to a significant increase in MDA content in callus tissue (58.3 vs 18.3 nmol / g), indicating severe oxidative damage to cells. This directly affected callus quality (score 2.6 vs 4.8) and embryogenic potential (42.3% vs 86.4%). This result demonstrates that the antioxidant protection system plays a crucial role in mitigating oxidative stress and improving callus quality.

[0076] 4. Importance of Osmotic Gradient Regulation (Comparative Example 4): Compared with a fixed sucrose concentration, osmotic gradient regulation significantly improved the induction rate of embryogenic callus (95.0% vs 74.6%) and embryogenic potential (86.4% vs 56.8%). This indicates that timely adjustment of the osmotic environment of the culture medium can effectively promote cell fate transition and embryogenic development.

[0077] 5. The effect of micronutrient enhancement (Comparative Example 5): Although the addition of micronutrient enhancers had little effect on the induction rate (95.0% vs 85.3%), it significantly improved callus quality (4.8 vs 4.1 points) and subculture stability (83.5% vs 62.8%). This indicates that micronutrients such as selenium, nickel, and silicon play an important role in maintaining cell viability and genetic stability.

[0078] The above comparative experimental results fully verify the scientific validity and effectiveness of the comprehensive technical solution of the three-stage progressive hormone regulation system combined with antioxidant protection and osmotic pressure gradient regulation of the present invention. Compared with the prior art, the method of the present invention shows significant advantages in terms of embryogenic callus induction rate, callus quality, embryogenic potential, and genetic stability, providing reliable technical support for somatic embryogenesis and plant regeneration in medium-grain coffee.

Claims

1. A method for inducing embryogenic callus in medium-sized coffee seeds, characterized in that, Includes the following steps: Medium-sized coffee leaves were selected as explants for surface sterilization. The sterilized explants were cultured in three stages: dedifferentiation, embryogenicity induction, and embryogenicity maintenance. The dedifferentiation period was carried out using a first culture medium supplemented with 2,4-dichlorophenoxyacetic acid, and cultured under the first culture conditions. The embryonic induction period was carried out using a second culture medium supplemented with 2,4-dichlorophenoxyacetic acid and 6-benzyladenine, and cultured under the second culture conditions. The embryogenicity maintenance period was carried out using a third culture medium supplemented with 1-naphthacetic acid and kinetin, and cultured under third culture conditions; Obtain embryogenic callus with firm texture and low degree of water immersion.

2. The method for inducing embryogenic callus in medium-sized coffee seeds according to claim 1, characterized in that, The first culture medium comprises the following components: Basic culture medium; 0.8-1.2 parts by weight of 2,4-dichlorophenoxyacetic acid; 25-35 parts by weight of sucrose; L-glutamine 3.5-4.5 parts by weight; Activated carbon 0.8-1.2 parts by weight; 6.5-7.5 parts by weight of agar.

3. The method for inducing callus formation in medium-sized coffee seeds according to claim 1, characterized in that, The second culture medium comprises the following components: Basic culture medium; 0.4-0.6 parts by weight of 2,4-dichlorophenoxyacetic acid; 6-Benzyladenine 2.0-2.5 parts by weight; 35-45 parts by weight of sucrose; Hydrolyzed casein 4.5-5.5 parts by weight; 4.5-5.5 parts by weight of L-proline; Ascorbic acid 0.9-1.1 parts by weight; Polyethylene glycol 4000: 3.5-4.5 parts by weight; Plant-based gel, 2.0-3.0 parts by weight.

4. The method for inducing callus formation in medium-sized coffee seeds according to claim 1, characterized in that, The third culture medium comprises the following components: Basic culture medium; 0.2-0.3 parts by weight of 1-naphthaleneacetic acid; Kinetin 0.9-1.1 parts by weight; 25-35 parts by weight of sucrose; 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; Plant-based gel, 2.0-3.0 parts by weight.

5. The method for inducing embryogenic callus in medium-sized coffee seeds according to any one of claims 1 to 4, characterized in that, The basal culture medium is a modified MS medium, comprising the following components: 15-20 parts by weight of ammonium nitrate; 18-22 parts by weight of potassium nitrate; Magnesium sulfate heptahydrate 3.5-4.5 parts by weight; 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, 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 callus formation in medium-sized coffee seeds according to claim 5, characterized in that, The basal culture 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 embryogenic callus in medium-sized coffee seeds according to claim 1, characterized in that, The first culture conditions were: temperature 25±1℃, cultured in complete darkness for 7-10 days, then switched to a 16 / 8 hour light / dark cycle, with a light intensity of... The pH value is 5.6-5.8, and the culture time is 21-28 days.

8. The method for inducing embryogenic callus in medium-sized coffee seeds according to claim 1, characterized in that, The second culture conditions were: temperature 26±1℃, 12 / 12 hour light / dark cycle, and light intensity... The pH value is 5.4-5.6, and the culture time is 14-21 days.

9. The method for inducing embryogenic callus in medium-sized coffee seeds according to claim 1, characterized in that, The third culture condition is: temperature 24±1℃, 16 / 8 hour light / dark cycle, light intensity... The pH value is 5.2-5.4, and the culture time is 21-28 days.

10. The method for inducing embryogenic callus in medium-sized coffee seeds 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; and washing with sterile water 5-6 times, 3-5 minutes each time.

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