Method for identifying cold resistance of coffee

Abstract

The invention relates to the technical field of plant germplasm resource identification, and discloses a method for identifying cold resistance of coffee. The identification method comprises the following steps: performing comprehensive cold resistance evaluation on the conductivity, catalase content, soluble sugar content and soluble protein content of obtained leaves by adopting coffee leaves subjected to low-temperature treatment through a membership function method to obtain cold resistance membership function values Uij of different coffee germplasm resources; and judging the cold resistance of the coffee resource through the obtained cold-resistant membership function value Uij. According to the method, a multi-index comprehensive evaluation system is established according to a membership function method, and the cold resistance of coffee can be effectively identified in the early stage. The identification method is suitable for a technical system for identifying and evaluating the early cold resistance of different coffee resources in the field in the natural cooling process, is matched with the winter field cold resistance identification result of a coffee planting area, and has important significance on utilization of different germplasm resources of coffee, reasonable planning and layout of a planting site and cold resistance breeding.

Description

Technical Field

[0001] The invention relates to the technical field of plant germplasm resource identification, and more specifically to a method for identifying cold resistance of coffee. Background Art

[0002] Coffee is the first of the three major beverage crops in the world. It is native to tropical regions and is suitable for growing in a climate environment with an average temperature of 18 to 25 degrees Celsius. The main coffee producing areas in China are Hainan and Yunnan. Hainan is mainly medium-grain coffee, and Yunnan is mainly small-grain coffee. The national coffee planting area is about 2.1 million mu, the output is 150,000 tons, and the total output value is about 2.1 billion yuan. Because my country's hot zone is located on the northern edge of the tropics, low temperatures often occur in winter, which usually limits the flowering and fruiting of coffee, causing the proportion of coffee beans to be too small, not full, and defective beans to increase, affecting the yield and quality of coffee, and in severe cases can lead to the death of the plant. Therefore, it is very important to improve the cold resistance of coffee and ensure that coffee can safely survive the winter.

[0003] Among them, breeding cold-resistant varieties and using coffee's own genetic factors to resist low temperature stress is the most fundamental way. The key to breeding cold-resistant varieties is the early identification of the cold resistance of germplasm resources. At present, the most reliable method to identify the cold resistance of coffee is direct identification. That is, when the test site encounters severe cold, on-site observation and comparative identification are carried out. However, direct identification is costly and is greatly restricted by the natural environment. In addition, direct observation often consumes a lot of manpower and cannot fully evaluate cold resistance.

[0004] Therefore, how to accurately and quickly identify the cold resistance of coffee germplasm resources at the seedling stage has become a technical problem that needs to be solved urgently in production. Summary of the invention

[0005] In view of this, the present invention is proposed. The present invention uses a multi-index comprehensive evaluation system and a membership function method to effectively identify and evaluate the cold resistance of coffee in an early stage, thereby improving accuracy.

[0006] In order to achieve the above object, the present invention provides the following technical solutions:

[0007] A method for identifying cold resistance of coffee, comprising:

[0008] Step 1: Obtain the second to fourth fully expanded mature leaves of the coffee seedlings; and subject to low temperature treatment at 8°C for 2-3 days.

[0009] Step 2, testing the leaves obtained in step 1 for four indicators: relative conductivity, catalase content, soluble sugar content, and soluble protein content; wherein the relative conductivity is calculated according to the inverse membership function Uij=1-(Xij-Xjmin) / (Xjmax-Xjmin) to calculate the cold resistance membership function value, and the catalase content, soluble sugar content, and soluble protein content are calculated according to the positive membership function Uij=(Xij-Xjmin) / (Xjmax-Xjmin) to calculate the cold resistance membership function value, and finally the average cold resistance membership function value is calculated as the average membership degree to identify the cold resistance of the coffee variety, and the higher the average membership degree, the higher the cold resistance of the coffee variety;

[0010] Uij represents the cold-resistance membership function value of the j index of the i variety; Xij represents the measured value of the j index of the i variety; Xjmin represents the minimum value of the j index of all varieties in the two groups; Xjmax represents the maximum value of the j index of all varieties in the two groups; i represents a certain variety; j represents a certain index.

[0011] The first leaf of the coffee seedling is relatively young, while the fourth and following leaves are too old. In order to reduce the error of the identification result, the second to fourth leaves are used in the present invention, which are mature and stable. In the present invention, the planting method of the coffee seedling is as follows:

[0012] Collect coffee berries, peel them and soak them in water, then degummed, washed and dried; the dried seeds are sown with sand and coconut bran as the substrate, and when the coffee seedlings grow to two leaves and one heart, they are bagged and transplanted.

[0013] Preferably, the relative conductivity of the present invention is measured by simple immersion or conventional extraction, and specific reference may be made to the literature "Comparative Study on the Determination Methods of Relative Conductivity of Plant Leaves" (Chen Aikui et al., Journal of Guangdong Education Institute, 2010). In a specific embodiment of the present invention, the relative conductivity detection method is as follows:

[0014] Wash the leaves, rinse with distilled water, rinse with deionized water, dry with filter paper, cut the leaves into pieces of 2 mm in length and width avoiding the veins, weigh 0.2 g, add 20 mL of deionized water, evacuate, pour into a conical flask and cover with a sealing film, oscillate at room temperature for 8 to 10 hours, oscillate at a frequency of 80 to 100 r / h, and then measure the conductivity R1; then seal the original solution and put it in a boiling water bath and boil for 30 to 35 minutes, cool to room temperature, measure the boiling conductivity R2 under the same oscillation conditions, repeat 3 times, and calculate the relative conductivity. Relative conductivity R (%) = (R1 / R2) × 100%.

[0015] In a specific embodiment of the present invention, catalase is determined using an (ammonium molybdate method) kit, soluble sugar content is determined using an (anthrone colorimetric method) kit, and soluble protein content is determined using a (spectrophotometric method) kit.

[0016] The present invention uses P4, P3, Caturra, Reyan 4, Reyan 1, 24-11, Dahan and Da7 as experimental materials, covering small-grain coffee, medium-grain coffee and large-grain coffee germplasm resources. Through the identification method of the present invention, it is identified that the cold resistance of the above-mentioned germplasm resources is P4>Caturra>P3>Dahan>24-11>Da7>Reyan 4>Reyan 1. The overall performance is that the cold resistance of small-grain coffee is stronger than that of large-grain and medium-grain coffee, which is consistent with the field cold resistance phenotype identification result in winter in coffee planting areas.

[0017] It can be seen from the above technical solutions that the technical effects achieved by the present invention are as follows:

[0018] The present invention measures the cold resistance only by using relevant parameters of leaves, and does not need to conduct experiments on the whole plant, thereby preventing the plant from dying in a cold environment. Moreover, the method can be scaled up and systematically identified, saving manpower.

[0019] The present invention uses the conductivity, catalase, soluble sugar content, and soluble protein content of coffee leaves at the seedling stage as indicators, and establishes a multi-index comprehensive evaluation system based on the membership function method, which can effectively identify and evaluate the cold resistance of coffee at an early stage. The coffee cold resistance identification method of the present invention is applicable to the technical system of early cold resistance identification and evaluation of different coffee resources in the field during the natural cooling process, and is consistent with the field cold resistance identification results in the coffee planting area in winter. The method is of great significance for the utilization of different varieties (germplasm) of coffee resources, the reasonable planning and layout of planting sites, and cold resistance breeding. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on the provided drawings without paying creative work.

[0021] Figure 1 Schematic diagram of the leaf sequence of a coffee seedling shown;

[0022] Figure 2 Shown is a comparative experiment on low temperature stress of large-grain coffee leaves, medium-grain coffee leaves and small-grain coffee leaves. DETAILED DESCRIPTION

[0023] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0024] The present invention discloses a method for identifying the cold resistance of coffee. Those skilled in the art can refer to the content of this article and appropriately improve the process parameters to achieve the method. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art and are considered to be included in the present invention. The identification method of the present invention has been described through preferred embodiments, and relevant personnel can obviously modify or appropriately change and combine the identification method described herein without departing from the content, spirit and scope of the present invention to implement and apply the technology of the present invention.

[0025] In a specific embodiment of the present invention, the coffee seedlings are cultivated in the following specific manner:

[0026] Collect coffee berries, peel them and soak them for 24 hours, remove the coffee seeds floating on the water, and then remove the remaining seeds, wash and dry them; spread the dried seeds on the sand bed and cover them with coconut bran, water them every day, and bag them when the coffee seedlings grow to two leaves and one heart. When the coffee grows to 6 months old, select the seedlings with normal growth, no pests and mechanical damage, and put them into an artificial incubator for 8℃ low temperature stress treatment.

[0027] The sand bed is firstly covered with sand of 15-20 cm, coffee seeds are evenly spread on the sand, a layer of fine sand of 1 cm thick is covered on the coffee seeds, coconut bran is covered on the sand, and a shade shed is built on the sand bed.

[0028] The present invention found that the relative permeability of the plasma membrane of the leaves reflects the degree of damage to the cell membrane of the leaves, and the relative permeability of the plasma membrane can be reflected by the conductivity; catalase is an antioxidant enzyme in coffee leaves, and the higher the enzyme activity, the stronger the ability to resist cold damage; soluble sugar and soluble protein are important osmotic regulating substances in coffee leaves, and the higher their content, the stronger the cold resistance of coffee. Therefore, the present invention selects these four indicators as the standards of the identification method of the present invention.

[0029] The following is a further description of the method for identifying the cold resistance of coffee provided by the present invention.

[0030] Example 1: Identification method of the present invention

[0031] 1. Cultivation and stress treatment of coffee seedlings

[0032] Collect coffee berries, peel them and soak them for 24 hours, remove and eliminate the coffee seeds floating on the water, then degummed the remaining seeds, washed and dried; spread the dried seeds on the sand bed and covered with coconut bran, watered every day, and bagged when the coffee seedlings grow to two leaves and one heart. When the coffee grows to 6 months old, select seedlings with normal growth, no pests and mechanical damage, and put them into an artificial incubator for 8℃ low temperature stress treatment for 2-3 days, and the untreated ones are used as the control.

[0033] 2. Determination of physiological and biochemical indicators of coffee leaves

[0034] Collect 2-3 fully expanded mature leaves from the second to the fourth leaf of coffee and number them respectively; measure the conductivity, catalase content, soluble sugar content, and soluble protein content of the leaves; set 3 repetitions for each sample. In order to describe the leaf sequence more vividly, the present invention provides Figure 1 The schematic diagram shown is used to demonstrate.

[0035] 3. Experimental data processing

[0036] The membership function method was used to evaluate the comprehensive cold resistance of the leaf conductivity, catalase, soluble sugar content, and soluble protein content obtained in step 2, and the cold resistance membership function values ​​U ij of different coffee varieties were obtained:

[0037] The indices positively correlated with cold resistance (catalase, soluble sugar content, and soluble protein content) were calculated using the positive membership function Uij = (Xij–Xjmin) / (Xjmax–Xjmin), and the indices negatively correlated with cold resistance (relative conductivity) were calculated using the inverse membership function Uij = 1–(Xij–Xjmin) / (Xjmax–Xjmin).

[0038] In the formula, Uij represents the cold resistance membership function value of the j index of the i variety; Xij represents the measured value of the j index of the i variety; Xjmin represents the minimum value of the j index of all varieties in the two groups; Xjmax represents the maximum value of the j index of all varieties in the two groups; i represents a certain variety; j represents a certain index.

[0039] The cold-resistance membership function value Uij of each indicator is obtained by calculation, and then the average cold-resistance membership function value is statistically calculated, which is called the average membership degree, so as to judge the cold-resistance of coffee. The larger the average membership degree, the stronger the cold-resistance of the variety.

[0040] Wherein, the conductivity is measured: wash the leaves, rinse with distilled water, rinse with deionized water, dry with filter paper, cut the leaves into pieces of 2 mm in length and width avoiding the veins, weigh 0.2 g, add 20 mL of deionized water, evacuate, pour into a conical flask and cover with a sealing film, oscillate at room temperature for 8 to 10 hours, the oscillation frequency is 80 to 100 r / h, and then measure the conductivity R1; then seal the original sample solution and put it in a boiling water bath and boil for 30 to 35 minutes, cool to room temperature, measure the boiling conductivity R2 under the same oscillation conditions, repeat 3 times, and calculate the relative conductivity. Relative conductivity R (%) = (R1 / R2) × 100%.

[0041] The catalase content was determined by a kit (ammonium molybdate method); the soluble sugar content was determined by a kit (anthrone colorimetric method); and the soluble protein content was determined by a kit (spectrophotometric method).

[0042] Example 2: Application of the identification method of the present invention

[0043] P4, P3, Kadura, Reyan 4, Reyan 1, 24-11, Dahan, and Da7 were selected as experimental materials and identified according to the method of Example 1. The results are shown in Tables 1 and 2.

[0044] Table 1 Index values ​​of different coffee germplasm resources and different groups

[0045]

[0046] Table 28℃ low temperature treatment of different coffee germplasm resources cold resistance membership function values ​​Uij

[0047]

[0048] Taking the P3 variety as an example, the calculation process of the cold resistance membership function value is demonstrated:

[0049] Conductivity Uij: 1-(87.0916-25.1516) / (120.7086-25.1516)=0.3518

[0050] Soluble sugar Uij: (156.9557-61.1593) / (171.2902-61.1593)=0.8698

[0051] Soluble protein Uij: (41.2959-21.8158) / (41.2959-21.8158)=1

[0052] CAT Uij:(10.9366-2.7209) / (12.8119-2.7209)=0.8142

[0053] Average Uij = (0.3518 + 0.8698 + 1 + 0.8142) / 4 = 0.7590

[0054] From Table 2, it can be determined that the cold resistance of the eight coffee resources is P4>Caturra>P3>Dahan>24-11>Da7>Reyan 4>Reyan 1. The overall performance is that the cold resistance of small-grain coffee is stronger than that of large-grain and medium-grain coffee, which is consistent with the existing field cold resistance phenotype identification results in winter in coffee planting areas, see the existing literature "Research on Domestic Coffee Photosynthesis" (Zhang Chuanzheng, Li Xinghui, Zhu Shigui; Tropical Agricultural Science and Technology, 2005) and "Coffee High-yield Cultivation Technology" (page 19, Dong Yunping, Long Yuzhou, Sun Yan, China Agricultural Press, 2009).

[0055] In addition, the present invention selected one of the above-mentioned test varieties, namely, small-grain coffee (P4), medium-grain coffee (Reyan 4) and large-grain coffee (Dahan), for low temperature stress test (leaf treatment was 8°C low temperature stress for 72h, and control was room temperature 25-27°C). The results are shown in FIG. Figure 2 ;

[0056] from Figure 2 From the performance point of view, the cold resistance is small-grain coffee (P4) > large-grain coffee (Daehan) > medium-grain coffee (Reyan No. 4), which is consistent with the results of the evaluation method of the present invention.

[0057] The method for identifying and evaluating the cold resistance of coffee of the present invention is applicable to a technical system for early identification and evaluation of the cold resistance of different coffee resources in the field during a natural cooling process. The cold resistance of winter coffee is identified by comprehensively using indicators such as the electrical conductivity and enzyme activity of the coffee, which is consistent with the results of field cold resistance identification in winter in coffee-growing areas, and can make early predictions for the winter cold resistance performance of coffee-growing areas.

[0058] The various embodiments in this specification are described in a progressive manner, and each embodiment focuses on the differences from other embodiments. The same or similar parts between the various embodiments can be referenced to each other.

[0059] The above description of the disclosed embodiments enables one skilled in the art to implement or use the present invention. Various modifications to these embodiments will be apparent to one skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention will not be limited to the embodiments shown herein, but rather to the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A method for identifying the cold resistance of coffee, characterized in that: include: Pick the second to fourth fully expanded and mature leaves of 6-month-old coffee seedlings of different varieties and treat them in an 8℃ environment for 2-3 days; Then, the cold resistance membership function value, catalase content, soluble sugar content and soluble protein content were calculated according to the inverse membership function Uij = 1-(Xij-Xjmin) / (Xjmax-Xjmin); the cold resistance membership function value was calculated according to the positive membership function Uij = (Xij-Xjmin) / (Xjmax-Xjmin), and finally the average cold resistance membership function value was statistically used as the average membership degree to identify the cold resistance of the coffee variety. The higher the average membership degree, the higher the cold resistance of the coffee variety. Among them, Uij represents the cold resistance membership function value of the j indicator of the i variety; Xij represents the measured value of the j indicator of the i variety; Xjmin represents the minimum value of the j indicator of all varieties in the two groups; Xjmax represents the maximum value of the j indicator of all varieties in the two groups; i represents a certain variety; j represents a certain indicator.

2. The identification method according to claim 1, characterized in that: The relative conductivity detection method adopts a simple immersion or conventional air extraction method for determination.

3. The identification method according to claim 1, characterized in that: The catalase assay is performed using the ammonium molybdate method.

4. The identification method according to claim 1, characterized in that: The soluble sugar content is determined by anthrone colorimetry.

5. The identification method according to claim 1, characterized in that: The soluble protein content was determined by spectrophotometry.

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