Method for breeding asexual high-yield coffee

Abstract

The invention relates to the technical field of plant breeding, in particular to an asexual high-yield coffee breeding method which comprises the following steps: screening superior plants to obtain a core stock plant, and preparing an asexual propagation material based on the core stock plant; carrying out seedling culture on the vegetative propagation material to obtain primary clonal seedlings, and carrying out seedling stage identification on the primary clonal seedlings to obtain healthy clonal seedlings; performing a field plot test on the healthy clonal seedlings to obtain candidate clones, and performing a multi-point regional test on the candidate clones to obtain stable clones; according to the method, asexual propagation technical parameters are optimized, composite hormone treatment is combined with a temperature control intermittent spraying technology for cutting seedling raising, and the rooting rate is larger than or equal to 80%; the tissue culture technology adopts an optimized culture medium formula, the propagation coefficient is greater than or equal to 5 times, the consistency is greater than or equal to 99%, and the problems of low propagation coefficient and low rooting survival rate of the traditional asexual propagation technology are solved.

Description

A method for breeding high-yield asexual coffee Technical Field

[0001] This invention relates to the field of plant breeding technology, and in particular to a method for breeding high-yield asexual coffee. Background Technology

[0002] Coffee, as one of the world's three major beverage crops, occupies an important position in tropical agricultural economies. In current coffee cultivation, medium-sized varieties (such as Robusta) are mostly cross-pollinated, and the offspring of seed propagation show significant phenotypic segregation, making it difficult to retain the superior traits of the mother plant; although small-sized varieties (such as Arabica) can self-pollinate, long-term planting easily leads to varietal degeneration, and the traditional sexual breeding cycle is as long as 25-30 years, resulting in extremely low breeding efficiency.

[0003] Existing asexual reproduction techniques, such as cuttings and grafting, suffer from low propagation coefficients and low rooting and survival rates. For example, cutting propagation relies on the upright branches of the mother plant, but the number of upright branches per coffee tree is limited, making it difficult to meet the demand for large-scale seedlings. Summary of the Invention

[0004] The purpose of this invention is to provide a method for breeding high-yield asexual coffee, which aims to solve the problems of low propagation coefficient and low rooting survival rate of traditional asexual reproduction technology.

[0005] To achieve the above objectives, this invention provides a method for breeding high-yield asexual coffee, comprising the following steps: selecting superior plants to obtain core mother plants, and preparing asexual propagation materials based on the core mother plants; cultivating the asexual propagation materials to obtain primary asexual seedlings, and identifying the primary asexual seedlings to obtain healthy asexual seedlings; conducting field plot trials on the healthy asexual seedlings to obtain candidate asexual lines, and conducting multi-point regional trials on the candidate asexual lines to obtain stable asexual lines; identifying the quality of the stable asexual lines to obtain high-quality stable asexual lines, and propagating them to obtain seedlings suitable for widespread distribution.

[0006] The target traits for selecting superior plants are: annual yield per plant ≥ 120% of the average of its population, which is stable for 3 consecutive years; susceptibility to coffee rust and leaf spot ≤ 5%; soluble solids content of small-bean coffee beans ≥ 18%; soluble solids content of medium-bean coffee beans ≥ 15%; and coffee bean size uniformity ≥ 90%.

[0007] The seedling cultivation process employs either cutting propagation or tissue culture. For cutting propagation, the substrate is a 1:1 volume ratio mixture of perlite and vermiculite. The environmental temperature is 25-29℃, the bottom temperature of the seedbed is 29℃, and the relative humidity is 90-95%. Intermittent spraying is used, and the rooting rate is ≥80% after 6-8 weeks. For tissue culture, fully expanded young leaves of the core mother plant serve as explants. The process involves callus induction, embryogenicity induction, somatic embryo formation, plant regeneration, and rooting hardening to obtain primary asexual seedlings with a uniformity ≥99%.

[0008] The seedling identification includes disease resistance identification, growth vigor identification, and morphological consistency identification. The disease resistance identification standard is a disease incidence rate of ≤3% after inoculation with coffee rust race. The growth vigor identification standard is a stem diameter of ≥0.3cm, a plant height of ≥10cm, and ≥6 pairs of leaves. The morphological consistency identification standard is that the seedling leaf shape and leaf color are consistent with the core mother plant.

[0009] The field plot experiment adopted a randomized block design, repeated 3 times, with the local main cultivated variety as the control, and was identified for 2 consecutive years. The clones with a total yield of ≥120% of the control and an interannual fluctuation of yield per plant ≤10% were selected as candidate clones.

[0010] Among them, the multi-point regional experiment sets up test sites in 3-5 different ecological type areas and identifies them for 3 consecutive years. The clones with annual yield per plant ≥ 110% of the control, annual yield fluctuation ≤ 8%, and survival rate ≥ 95% at all test sites are considered stable clones.

[0011] The quality assessment includes GC-MS volatile component analysis, high-performance liquid chromatography caffeine content determination, and cupping score, with the total content of characteristic flavor components ≥0.8mg / g and the cupping score ≥80 points.

[0012] This invention discloses a method for breeding high-yield asexual coffee plants. The method involves selecting superior plants to obtain core mother plants, and preparing asexual propagation materials based on these core mother plants. The asexual propagation materials are then cultivated to obtain primary asexual seedlings, which are then assessed at the seedling stage to obtain healthy asexual seedlings. These healthy seedlings are then subjected to field plot trials to obtain candidate asexual lines, which are then subjected to multi-location regional trials to obtain stable asexual lines. Finally, the stable asexual lines are assessed for quality to obtain high-quality stable asexual lines, which are then propagated to obtain... From core mother plant selection to final seedlings suitable for widespread cultivation, this method employs a progressive screening and identification process. Each step, from core mother plant selection to final seedlings suitable for widespread cultivation, has strict evaluation criteria to ensure that the selected coffee clones possess stable high-yield, high-quality, disease-resistant, and widely adaptable traits. The method optimizes asexual reproduction technical parameters. Cutting propagation uses compound hormone treatment combined with temperature-controlled intermittent spraying technology, achieving a rooting rate of ≥80%. Tissue culture technology uses an optimized culture medium formula, with a proliferation coefficient of ≥5 times and a uniformity of ≥99%, thus solving the problems of low propagation coefficient and low rooting survival rate in traditional asexual reproduction techniques. Attached Figure Description

[0013] To more clearly illustrate the technical solutions in the embodiments of this application or the prior art, the accompanying drawings used in the description of the embodiments or the prior art will be briefly introduced below.

[0014] The terms "first," "second," "third," and "fourth," etc., used in the specification, claims, and accompanying drawings of this application are used to distinguish different objects, not to describe a specific order. Furthermore, the terms "comprising" and "having," and any variations thereof, are intended to cover non-exclusive inclusion. For example, a process, method, system, product, or apparatus that includes a series of steps or units is not limited to the listed steps or units, but may optionally include steps or units not listed, or may optionally include other steps or units inherent to these processes, methods, products, or apparatuses.

[0015] It should be noted that all information (including but not limited to user device information, user personal information, etc.), data (including but not limited to data used for analysis, stored data, displayed data, etc.), and signals involved in this application have been authorized by the user or fully authorized by all parties, and the collection, use, and processing of related data must comply with the relevant laws, regulations, and standards of the relevant countries and regions. For example, the strain data, acceleration data, displacement data, pressure data, and video data involved in this application were all obtained with full authorization.

[0016] Figure 1 is a flowchart of a method for breeding high-yield asexual coffee plants provided by the present invention. Detailed Implementation

[0017] The embodiments of the present invention are described in detail below. Examples of the embodiments are shown in the accompanying drawings. The embodiments described below with reference to the accompanying drawings are exemplary and intended to explain the present invention, but should not be construed as limiting the present invention.

[0018] Please refer to Figure 1. The present invention provides a method for breeding high-yield asexual coffee plants, which includes the following steps: S1 Selecting superior plants to obtain core mother plants, and preparing asexual propagation materials based on the core mother plants; In the embodiment of the present invention, in the mature plantations (trees aged 5-8 years) in the core coffee producing area, areas with consistent planting density and the same cultivation and management conditions are selected as screening nurseries. The target traits for selecting superior plants were set as follows: annual yield per plant ≥ 120% of the population average, stable for 3 consecutive years; susceptibility to coffee rust and leaf spot ≤ 5%; soluble solids content ≥ 18% for small-bean coffee and ≥ 15% for medium-bean coffee; coffee bean size uniformity ≥ 90%; compact tree shape; plant height ≤ 2.5m; branching angle 30-45°. Coffee trees in the selection nursery were marked, and the yield per plant was measured at each harvest season for 3 consecutive years. The disease resistance and agronomic traits of the plants were recorded. Plants with annual yield fluctuations exceeding 20%, susceptibility > 5%, or agronomic traits that did not meet the requirements were removed. Candidate superior plants that met the initial screening criteria were retained. The coffee beans of the candidate superior plants were analyzed for quality. Soluble solids content was measured using a refractometer, and coffee bean size uniformity was measured by sieving. Plants that did not meet the quality indicators were removed. After the above screening process, the plants that met all the indicators were selected as core mother plants.

[0019] Select healthy, vigorous, green, upright shoots from February to March, 15-20 days after the core mother plant's new shoots emerge. Choose 2-3 sections of these shoots below the terminal bud, ensuring they are free from pests and diseases. Cut these branches into 4-6cm single-node cuttings, retaining one plump axillary bud per node. Remove the lower leaves and trim the upper leaves to 6-8cm. 2 (Retain 1 / 3 of the leaf area), cut the lower end of the cutting at an angle (45°, with a flat cut surface), and immediately soak it in clean water for 30 minutes. After soaking, take out the cutting, drain the water, and dip the lower cut surface into a compound hormone solution (1000ppm IBA + 500ppm NAA). Dip it quickly for 1-2 seconds, and then gently tap off the excess solution to obtain asexual propagation material for cuttings.

[0020] S2 involves cultivating the asexual propagation material to obtain primary asexual seedlings, and then identifying the primary asexual seedlings to obtain healthy asexual seedlings. In this embodiment of the invention, the seedling cultivation adopts cutting propagation or tissue culture. The substrate for cutting propagation is a mixture of perlite and vermiculite in a 1:1 volume ratio. The seedling environment temperature is 25-29℃, the bottom temperature of the seedbed is 29℃, the relative humidity is 90-95%, and intermittent spraying is used. After 6-8 weeks, the rooting rate is ≥80%. The tissue culture seedlings use the stem tip or embryonic embryo of the core mother plant as explants. After callus induction, embryogenicity induction, regeneration, and rooting hardening, primary asexual seedlings are obtained with a uniformity of ≥99%.

[0021] Taking cutting propagation as an example, perlite and vermiculite are mixed in a 1:1 volume ratio, and carbendazim wettable powder (concentration 500ppm) is added for disinfection. After mixing, the mixture is placed in seedling trays (5cm aperture, 10cm depth). The cuttings from the asexual propagation material are inserted into the seedling substrate to a depth of 1 / 2-2 / 3 of the cutting length. The substrate is compacted, and the trays are thoroughly watered. The seedling trays are then placed in a greenhouse, with the temperature controlled at 25-29℃. A heating device is used at the bottom of the seedbed to maintain a temperature of 29℃ and a relative humidity of 90-95%. Intermittent spraying is employed. The misting system should be used, spraying once every 30 minutes during the day for 30 seconds each time, and stopping spraying at night. The light intensity should be controlled at 2000-3000 lux, with a light duration of 12 hours / day. Starting from the 15th day after cutting, apply foliar fertilizer (containing 10% nitrogen, 5% phosphorus, and 8% potassium) once a week at a concentration of 0.2% for 4 weeks. Diseased cuttings should be removed promptly to prevent the spread of disease. 6-8 weeks after cutting, when the rooting rate of the cuttings is ≥80%, the length of the new roots is ≥3cm, and the length of the sprouted new shoots is ≥2cm, they are considered the first generation of asexual seedlings.

[0022] The seedling identification includes disease resistance identification, growth vigor identification, and morphological consistency identification. The disease resistance identification standard is a disease incidence rate of ≤3% after inoculation with coffee rust race. The growth vigor identification standard is a stem diameter of ≥0.3cm, a plant height of ≥10cm, and ≥6 pairs of leaves. The morphological consistency identification standard is that the seedling leaf shape and leaf color are consistent with the core mother plant.

[0023] Disease resistance identification: The artificial inoculation method was used, and a suspension of coffee rust spores (concentration 1×10⁻⁶) was inoculated. 6The solution (number of seeds / mL) was evenly sprayed onto the leaf surface of the first-generation clonal seedlings and placed in an incubator with 95% humidity and 25℃ for 7 days. The disease susceptibility was observed and recorded, and seedlings with a disease susceptibility rate >3% were removed. Growth vigor assessment: For seedlings that passed the disease resistance assessment, the stem diameter (2cm from the base), plant height, and number of leaves were measured, and seedlings that did not meet the growth vigor indicators were removed. Morphological consistency assessment: The leaf shape and color of the seedlings were observed and compared with the leaf morphology of the core mother plant, and seedlings with significant morphological differences were removed. After the above assessment, the seedlings that met all the indicators were considered healthy clonal seedlings.

[0024] S3. Field plot trials are conducted on the healthy clonal seedlings to obtain candidate clonal lines. Multi-site regional trials are then conducted on the candidate clonal lines to obtain stable clonal lines. In this embodiment of the invention, the field plot trials employ a randomized block design, repeated three times, with the local main cultivated variety as a control. The trials are conducted for two consecutive years. Clonal lines with a total yield ≥ 120% of the control and an annual yield fluctuation of ≤ 10% per plant are considered candidate clonal lines. The multi-site regional trials are conducted at 3-5 different ecological zones, with three consecutive years of evaluation. Clonal lines with an annual yield of ≥ 110% of the control at all test sites, an annual yield fluctuation of ≤ 8%, and a survival rate ≥ 95% are considered stable clonal lines.

[0025] A randomized block design with three replicates was used. Each plot was 20 m² (4 m × 5 m) with a plant spacing of 2 m × 1.5 m, and six healthy clonal seedlings were planted in each plot. The local main coffee variety was used as the control (CK), and the cultivation and management conditions of the control plots were the same as those of the experimental plots. The experimental site was selected in an area with similar ecological conditions to the screening nursery (average annual temperature 22-25℃, altitude 800-1000 m), with lateritic soil type, pH 5.5-6.5, and organic matter content ≥2%. Conventional coffee cultivation and management techniques were followed, including balanced fertilization, integrated pest management, and pruning (pruning once a year in winter to remove diseased, weak, and overly dense branches). Evaluation was conducted for two consecutive years, and agronomic traits such as total yield, average yield per plant, plant height, number of branches, and number of fruiting branches were recorded for each plot. Quality indicators such as soluble solids content and size uniformity of coffee beans were measured. The occurrence of pests and diseases was also recorded. A clone that has a total yield of ≥120% of the total yield of the control clone, an annual fluctuation of yield per plant of ≤10%, meets the quality indicators set by S1, and has a disease and pest susceptibility rate of ≤5% is considered a candidate clone.

[0026] Three to five experimental sites were set up in different ecological zones, covering low (600-800m), medium (800-1000m), and high (1000-1200m) altitude areas. Each experimental site had representative soil type and climate conditions, and consistent cultivation and management conditions. A randomized block design was used for each experimental site, with three replicates and a plot area of ​​30m².2 The plant spacing was 2m × 1.5m, with 10 plants per plot (5m × 6m) and 5 plants per plot (candidate clones). The local main cultivar was used as the control. The experiment was conducted for three consecutive years, recording yield data (total yield per plot, yield per plant), agronomic traits (plant height, number of branches, fruiting habits), disease resistance (susceptibility rate under natural disease conditions), and adaptability (survival rate, growth vigor) at each experimental site annually. A stable clone was defined as one whose annual yield per plant was ≥ 110% of the control variety, whose annual yield fluctuation was ≤ 8%, whose disease resistance and susceptibility rate were ≤ 5%, whose survival rate was ≥ 95%, and whose agronomic traits were stable.

[0027] S4 performs quality assessment on the stable clones to obtain high-quality stable clones, and then propagates and cultivates them to obtain seedlings that can be promoted.

[0028] In this embodiment of the invention, the quality assessment includes GC-MS volatile component analysis, high-performance liquid chromatography caffeine content determination, and cupping score, with the total content of characteristic flavor components ≥0.8mg / g and the cupping score ≥80 points.

[0029] Specifically, the coffee beans harvested from each test site were hulled and dried (moisture content reduced to 10-12%), then ground into coffee powder. GC-MS was used to analyze the volatile components of the coffee powder, requiring a total content of characteristic flavor components such as furans and pyrazines ≥0.8 mg / g. High-performance liquid chromatography (HPLC) was used to determine the caffeine content, requiring 0.8-1.2% caffeine for small-bean coffee and 1.8-2.2% caffeine for medium-bean coffee. Five professional tasters scored the coffee according to international cupping standards, including aroma, flavor, acidity, body, and finish. A total score ≥80 points was required, and the clonal lines meeting all the above quality indicators were considered high-quality stable clonal lines.

[0030] Healthy branches from high-quality, stable clones were selected as propagation materials, with priority given to upright branches grown in February and March to ensure the phenotypic stability of the propagated offspring. Large-scale propagation was carried out using either cuttings or tissue culture, with simultaneous seedling cultivation under propagation conditions. The rooting rate of cuttings was ≥85%, and the proliferation coefficient of tissue culture propagation was ≥5 times. The propagated seedlings were planted to establish a primary seed nursery. The nursery site must be located far from other coffee plantations (isolation distance ≥500m) to prevent pollen contamination. The cultivation and management conditions in the primary seed nursery were superior to those in conventional plantations, with regular pest and disease monitoring and control. Every 3 years, the phenotypic characteristics of the plants in the primary seed nursery were reviewed, and mutant plants were removed. Select healthy, disease-free seedlings with traits consistent with the high-quality stable clones from the original seed nursery as the final product (seedlings that can be promoted). The seedling standards are: seedling age 6-8 months, stem diameter ≥0.5cm, plant height ≥20cm, and well-developed root system (main root length ≥5cm, number of lateral roots ≥10).

[0031] The above-disclosed embodiments are merely preferred embodiments of asexual high-yield coffee breeding methods of this application and should not be construed as limiting the scope of this application. Those skilled in the art can understand that all or part of the processes of the above embodiments can be implemented, and equivalent changes made in accordance with the claims of this application still fall within the scope of this application.

Claims

1. A method for breeding high-yield asexual coffee, characterized in that... The process includes the following steps: selecting superior plants to obtain core mother plants, and preparing asexual propagation materials based on the core mother plants; cultivating the asexual propagation materials to obtain primary asexual seedlings, and identifying the primary asexual seedlings to obtain healthy asexual seedlings; conducting field plot trials on the healthy asexual seedlings to obtain candidate asexual lines, and conducting multi-point regional trials on the candidate asexual lines to obtain stable asexual lines; identifying the quality of the stable asexual lines to obtain high-quality stable asexual lines, and propagating them to obtain seedlings suitable for widespread distribution.

2. The method for breeding high-yield asexual coffee as described in claim 1, characterized in that... The target traits for selecting superior plants are: annual yield per plant ≥ 120% of the average of its population, which is stable for 3 consecutive years; susceptibility to coffee rust and leaf spot ≤ 5%; soluble solids content of small-bean coffee beans ≥ 18%; soluble solids content of medium-bean coffee beans ≥ 15%; and coffee bean size uniformity ≥ 90%.

3. The method for breeding high-yield asexual coffee as described in claim 1, characterized in that... The seedling cultivation adopts cutting propagation or tissue culture. The substrate for cutting propagation is a mixture of perlite and vermiculite in a 1:1 volume ratio. The seedling environment temperature is 25-29℃, the bottom temperature of the seedbed is 29℃, the relative humidity is 90-95%, and intermittent spraying is used. After 6-8 weeks, the rooting rate is ≥80%. The tissue culture seedling uses the fully expanded tender leaves of the core mother plant as explants. After callus induction, embryogenicity induction, somatic embryo formation, plant regeneration, and rooting hardening, the first generation of asexual seedlings are obtained with a uniformity of ≥99%.

4. The method for breeding high-yield asexual coffee as described in claim 1, characterized in that... The seedling identification includes disease resistance identification, growth vigor identification, and morphological consistency identification. The disease resistance identification standard is a disease incidence rate of ≤3% after inoculation with coffee rust race. The growth vigor identification standard is a stem diameter of ≥0.3cm, a plant height of ≥10cm, and ≥6 pairs of leaves. The morphological consistency identification standard is that the seedling leaf shape and leaf color are consistent with the core mother plant.

5. The method for breeding high-yield asexual coffee as described in claim 1, characterized in that... The field plot experiment adopted a randomized block design, repeated 3 times, with the local main cultivated variety as the control, and was identified for 2 consecutive years. The clones with a total yield of ≥120% of the control and an interannual fluctuation of yield per plant ≤10% were selected as candidate clones.

6. The method for breeding high-yield asexual coffee as described in claim 1, characterized in that... The multi-point regional experiment sets up test sites in 3-5 different ecological types and evaluates them for 3 consecutive years. The clones with annual yield per plant ≥ 110% of the control, annual yield fluctuation ≤ 8%, and survival rate ≥ 95% at all test sites are considered stable clones.

7. The method for breeding high-yield asexual coffee as described in claim 1, characterized in that... The quality assessment includes GC-MS volatile component analysis, high-performance liquid chromatography caffeine content determination, and cupping score. The total content of characteristic flavor components is ≥0.8mg / g, and the cupping score is ≥80 points.

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