A method for identifying and screening cold tolerance of pennisetum and application thereof

Abstract

The application provides a method for identifying and screening cold tolerance of elephant grass and application thereof, and relates to the field of plant cultivation. The method mainly comprises the following steps: subjecting the standard elephant grass stem segments to variable temperature germination; subjecting the standard elephant grass stem segments to constant temperature low temperature stress; subjecting the stressed stem segments to recovery culture under suitable conditions; determining and calculating the low temperature germination, stress survival rate and recovery growth rate of the stem segments; and comprehensively evaluating the cold tolerance of the stem segments based on the weighted values of the three. The application overcomes the shortcomings of the prior art, can simulate the key low temperature stress and recovery process in the laboratory, and can realize rapid, reliable and batch preliminary identification and screening of the cold tolerance of a large amount of materials, so that the blind dependence on large-scale and long-period field identification is significantly reduced, the screening accuracy and breeding efficiency are effectively improved, and the breeding period is shortened.

Description

Technical Field

[0001] This invention relates to the field of plant cultivation, specifically to a method for identifying and screening the cold resistance of elephant grass and its application. Background Technology

[0002] Elephant grass (Pennisetum purpureum), native to tropical Africa, is an important forage grass widely cultivated in tropical and subtropical regions worldwide. A perennial, tall grass with high photosynthetic efficiency, elephant grass offers significant advantages in high yield and low cost in the roughage production system for cattle and sheep intensive farming in southern my country. Due to its tropical origin, elephant grass's inherent warm-loving characteristics result in a very weak natural cold-resistant genetic base within its germplasm resources. The limited number of elephant grass germplasm resources introduced to my country makes traditional hybridization methods for improving cold-resistant varieties challenging, as they face bottlenecks such as narrow parental selection, low genetic gain, long breeding cycles, and limited breakthroughs. While modern molecular breeding techniques such as gene editing are theoretically precise, the gene network regulation mechanism of cold resistance in elephant grass, a complex polyploid non-model crop, remains unclear, and related technical systems are far from mature. Therefore, in current practice, relying on natural stalk and shoot variation and physical mutagenesis remains the main approach for cultivating new cold-resistant lines. This approach suffers from inherent drawbacks such as a large quantity of field identification materials, low identification efficiency, and a long continuous cycle. More importantly, cold tolerance, as a complex quantitative trait controlled by multiple genes, is heavily influenced by the interaction between genotype and environment. Current identification techniques largely rely on the low-temperature half-lethal temperature (LT). 50 The laboratory also measures in vitro physiological and biochemical indicators such as malondialdehyde (MDA), peroxidase (POD), catalase (CAT) activity, and soluble sugar content. However, these indicators typically only reflect the plant's stress response at a specific moment or in a short period, and have limited correlation with the plant's overall cold resistance (such as survival rate, greening rate, and growth recovery ability) throughout the entire field overwintering cycle. This disconnect between "laboratory indicators" and "field performance" makes the efficient, large-scale screening of elephant grass cold-resistant materials in the laboratory extremely unreliable.

[0003] As a perennial asexually propagated forage crop, the cold resistance of near-ground stem shoots is crucial for overwintering, greening, and forage yield in the following year. However, a mature and unified technical standard and procedure for identifying this trait has not yet been established in China. Currently, related research and breeding practices mainly refer to other gramineous crops with similar growth habits and reproductive methods, with sugarcane having the most mature identification system. A national recommended standard for sugarcane, "Technical Procedure for Identification of Sugarcane Cold Resistance" (GB / T35836-2018), has been established. This standard systematically specifies the methods, investigation items, and evaluation system for field identification, providing important support for the breeding of new cold-resistant varieties. Its overall research approach has general methodological reference value for plants like elephant grass that reproduce via stem nodes. However, directly applying this standard to the identification of elephant grass cold resistance presents a fundamental technical mismatch, mainly in the following two aspects:

[0004] (1) The essential differences between breeding objectives and core evaluation indicators

[0005] Sugarcane, an annual or perennial crop whose economic product is its stalks, is evaluated for its cold resistance primarily by quantifying the direct economic losses caused by low temperatures to the processing quality of the stalks. Elephant grass, on the other hand, is a perennial forage crop with an annual biomass production target. Its cold resistance development focuses on ensuring the efficient survival and rapid recovery of underground rhizomes and near-ground dormant buds after winter (i.e., regreening and sprouting). The fundamental differences between sugarcane and elephant grass in terms of economic attributes, production cycles, and breeding objectives determine that the sugarcane cold resistance evaluation standard is unsuitable for the elephant grass cold resistance evaluation, which focuses on "winter survival rate" and "stalk regeneration ability."

[0006] (2) Inherent technical defects of relying on natural field identification

[0007] From a breeding application perspective, current evaluations of elephant grass cold hardiness still rely on phenotypic observations of natural overwintering in the field. This method has the following drawbacks: First, the timing, intensity, duration, and cooling pattern of natural low-temperature events are uncontrollable, and low-temperature stress often interacts with complex environmental factors such as soil moisture, microbial activity, and pests and diseases, resulting in poor experimental repeatability and high randomness. Second, to eliminate the interference of annual climate fluctuations and differences in plot microenvironments, reliable conclusions must be obtained through repeated experiments over many years and at multiple locations, resulting in a long identification cycle (often measured in years), high labor and land costs, and low screening efficiency, which seriously restricts the breeding process.

[0008] In summary, there are clear technological gaps in the existing technology: on the one hand, there is a lack of a standardized indoor identification method that can overcome the limitations of field identification and rapidly, accurately, and in large quantities assess the cold resistance of elephant grass stem shoots under controlled conditions; on the other hand, there is also a lack of evaluation standards and indicator systems specifically targeting the cold resistance biological objective of elephant grass, namely "overwintering survival-greening and sprouting". This technological bottleneck has become a key obstacle restricting the efficient screening of cold-resistant germplasm resources of elephant grass and the breakthrough in the breeding of new varieties. Summary of the Invention

[0009] To address the shortcomings of existing technologies, this invention provides a method for identifying and screening the cold resistance of elephant grass and its application. By simulating key low-temperature stress and recovery processes in the laboratory, it enables rapid, reliable, and large-scale preliminary identification and screening of the cold resistance (especially the survival and germination potential of stem buds) of a large number of materials. This significantly reduces the blind reliance on large-scale, long-term field identification, effectively improves screening accuracy and breeding efficiency, and shortens the breeding cycle.

[0010] To achieve the above objectives, the present invention provides the following technical solution:

[0011] A method for identifying and screening the cold resistance of elephant grass, the method comprising the following steps:

[0012] S0. Several elephant grass materials to be identified were asexually propagated to form a certain number of populations to meet the experimental sampling requirements;

[0013] S1. Take a stem segment containing a single dormant bud from the second to last yellow leaf to the third node from the base of an elephant grass plant that was asexually propagated before overwintering as the identification material.

[0014] S2. The above-mentioned identification materials were evenly divided into Experimental Group 1, Control Group 1, Experimental Group 2, and Control Group 2.

[0015] The identification materials of the experimental group were hydroponically cultured for 7-14 days under a variable temperature mode of 15℃, 16h and 8℃, 8h; the identification materials of the control group were hydroponically cultured for 7-14 days under a constant temperature mode of 20-25℃.

[0016] The identification materials of Experimental Group 2 were placed at a low temperature of 4±0.5℃ for 4 hours, then transferred to a constant temperature of -1℃ to -3℃ for 60-84 hours, then placed at a low temperature of 4±0.5℃ for 6-8 hours, and then hydroponically treated for 7-14 days; the identification materials of Control Group 2 were placed at a constant temperature of 20-25℃ for the same time as Experimental Group 2, and then hydroponically treated at 20-25℃ for 7-14 days.

[0017] S3. Record the root germination rate of experimental group 1 as the low-temperature root germination rate, and the root germination rate of control group 1 as the control root germination rate; the average shoot length of experimental group 1 is the low-temperature average shoot length, and the average shoot length of control group 1 is the control average shoot length; calculate the root sprouting ability, bud sprouting ability, and low-temperature germination ability of experimental group 1, where root sprouting ability = low-temperature root germination rate / control root germination rate; bud sprouting ability = low-temperature average shoot length / control average shoot length; low-temperature germination ability = root sprouting rate × 0.6 + bud sprouting ability × 0.4.

[0018] S4. Record the root germination rate, average root length, seedling survival rate, seedling height, and number of leaves for experimental group 2 and control group 2; calculate the stress survival rate and recovery rate of experimental group 2, where the stress survival rate = (root germination rate of experimental group 2 / root germination rate of control group 2) × 0.6 + (seedling survival rate of experimental group 2 / seedling survival rate of control group 2) × 0.4; recovery rate = ((root length of experimental group 2 / root length of control group 2) + (seedling height of experimental group 2 / seedling height of control group 2) + (number of leaves of experimental group 2 / number of leaves of control group 2)) ÷ 3;

[0019] S5. Calculate the cold resistance index of the identified materials based on the above-mentioned low-temperature germination rate, stress survival rate, and recovery rate. The cold resistance index is calculated as follows: (low-temperature germination rate × 0.3 + stress survival rate × 0.3 + recovery growth rate × 0.4). Materials with a cold resistance index ≥ 0.7 are considered strongly cold-resistant, those with a cold resistance index between 0.7 and 0.5 are considered weakly cold-resistant, and those with a cold resistance index < 0.5 are considered non-cold-resistant.

[0020] Preferably, in step S1, the cut of the material being identified is at a 45° angle, with a length of 3 cm below the segment and 5 cm above the segment.

[0021] Preferably, in step S1, when there is insufficient identification material obtained from the same plant of elephant grass, the material is taken from the third green leaf upwards.

[0022] Preferably, in step S2, the identification materials of both experimental group 2 and control group 2 are subjected to constant temperature treatment in a dry state.

[0023] Preferably, in step S2, the hydroponic treatment temperature for experimental group 2 and control group 2 for 12 days is room temperature 20-25℃.

[0024] The above method is applied to the cultivation of new cold-resistant elephant grass varieties, and the application method includes the following steps:

[0025] (1) Elephant grass seedlings with a cold hardiness index of 0.7 or above were temporarily planted in a greenhouse and transplanted to an altitude of 2000m in mid-to-late March. The rooting rate and plant growth rate were counted 30 days after the greening up the following year, and the cold hardiness overwintering ability was calculated. The cold hardiness overwintering ability = rooting rate × 0.4 + plant growth rate × 0.6;

[0026] (2) Select materials with cold resistance to winter ≥0.8 or individual data ≥1.0 to establish clones as materials for subsequent new line breeding.

[0027] Preferably, the greening time is defined as the period during which at least 30% of the plant clumps have at least one new sprout.

[0028] Preferably, the germination rate = number of germinations / total number of statistics; the plant growth rate = number of sprouts / number of stubble.

[0029] This invention provides a method for identifying and screening the cold resistance of elephant grass and its application, which has the following advantages compared with the prior art:

[0030] (1) This invention sets up three quantitative indicators: “low-temperature germination,” “stress survival rate,” and “growth recovery rate,” and integrates them into a unified “cold resistance” index through a scientific weighted formula. This indicator system overcomes the limitations of traditional reliance on single, instantaneous physiological indicators (such as electrolyte leakage rate) or subjective morphological descriptions. It can comprehensively and objectively reflect the entire process of materials from low-temperature germination potential and stress tolerance to recovery and regeneration ability after damage, enabling accurate comparison and ranking of the cold resistance of materials with different genetic backgrounds, and greatly improving the accuracy and comparability of identification.

[0031] (2) This invention compresses the field wintering evaluation, which takes several years, into a standardized indoor identification process that can be completed within a few weeks. Through indoor identification, materials with strong cold resistance potential (cold resistance ≥0.7) can be quickly screened from a large number of germplasms, and only these selected materials are put into high-altitude field verification. This strategy reduces the scale and workload of field trials by more than 70%, shortens the preliminary screening cycle from the traditional 1-2 wintering years to a few weeks, and shortens the overall breeding cycle by 1-2 years, significantly reducing the input of human, material and land resources, and greatly improving breeding efficiency;

[0032] (3) The scheme takes "growth recovery ability" as the core evaluation criterion, ensuring a high degree of consistency between the identification results and the final breeding goals. This scheme assigns the highest weight (0.4) to "growth recovery rate" and designs a detailed hydroponic recovery observation process, which directly simulates and quantifies the key agronomic trait of "greening and sprouting" after overwintering of elephant grass. It achieves a high degree of fit between the indoor identification results and the actual overwintering survival and regeneration performance in the field, fundamentally solving the core technical problem of the disconnect between existing laboratory indicators and field performance;

[0033] (4) The scheme makes clear and specific provisions for every aspect, including material specifications, treatment temperature and duration, control settings, index measurement and calculation formulas, forming a standardized process that can be repeated and operated in batches. This lays a solid technical foundation for the formulation of the first industry or local standard in the field of elephant grass cold resistance identification, and is conducive to achieving technical unification and resource sharing in this field. Attached Figure Description

[0034] Figure 1 This is a complete schematic diagram of the segmentation of the elephant grass seed stem according to the present invention. The diagram shows the continuous segmentation of the elephant grass seed stem from bottom to top from right to left. According to the number of nodes of the elephant grass seed stem from bottom to top, the buds on nodes 1-7 are strong buds (dormant strong buds), the buds on nodes 8-11 are sprouting buds, the buds on node 12 are dormant weak buds, and the buds on nodes 13-15 are sprouting buds.

[0035] Figure 2 This is a schematic diagram illustrating the characteristics of robust buds, insect-damaged buds, sprouting buds, and germinating buds in the experimental process of this invention.

[0036] Figure 3 This is a schematic diagram showing the changes in germination rate of RM-1 at different locations after different water loss times of 0, 5, and 10 days during the experimental process of this invention.

[0037] Figure 4 This is a schematic diagram showing the changes in germination rate of RY-4 at different locations after different water loss times of 0, 5, and 10 days during the experimental process of this invention.

[0038] Figure 5 This is a schematic diagram showing the relationship between the duration of -2℃ and the survival rate of buds and roots of material RY-4 in the temperature and time selection experiment for evaluating the cold resistance of elephant grass in this invention.

[0039] Figure 6 This is a schematic diagram showing the relationship between the duration of -3℃ and the survival rate (both root and bud sprouting) of seedlings of RY-4 (weakly cold-resistant) and No. 6 (strongly cold-resistant) materials in the temperature and time selection experiment for evaluating the cold resistance of elephant grass in this invention.

[0040] Figure 7 The growth of moderately cold-resistant materials (Table 3, No. 8) after hydroponics at -2℃ for different durations of 12 days (top left to right: hydroponics results for -2℃ stress times of 0h, 36h, 48h, and 60h; bottom left to right: hydroponics results for -2℃ stress times of 72h, 84h, and 96h; bottom right: materials with -2℃ stress time of 120h, no obvious sprouting of stem nodes).

[0041] Figure 8 This is a schematic diagram comparing the frost damage to the stem tips of the seven highly cold-resistant materials screened in the laboratory during late autumn and early winter with the frost damage of the control material RY-4 (left 1-7 are the seven highly cold-resistant materials screened, numbered YX2401, YX2402, YX2403, YX2404, YX2405, YX2406, YX2407 respectively, left 8 is the control material RY-4).

[0042] Figure 9The comparison of frost damage to stems and buds in this invention is shown (left 1 is the highly cold-resistant YX25-48 obtained by screening overwintering stems and buds, left 2 and left 3 are cross-sectional and surface diagrams of frost damage to buds of RY-4).

[0043] Figure 10 This is a schematic diagram illustrating the difference in growth between strongly cold-resistant (red line) and weakly cold-resistant (yellow line) materials after overwintering and regrowth in the experiment of this invention;

[0044] Figure 11 A schematic diagram showing the differences in hydroponic growth of different RY-4 plants after natural overwintering stem shoots under low-temperature stress.

[0045] Figure 12 The experiment of this invention compares the greening of the highly cold-resistant material YX25-48 and the control material RY-4, with the highly cold-resistant material on the right.

[0046] Figure 13 This is a schematic diagram showing the growth of the cold-resistant material YX25-48 and the control material RY-4 after they turned green in the invention experiment. The cold-resistant material is shown at the bottom. Detailed Implementation

[0047] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0048] Elephant grass reproduces asexually, and several plants can be propagated in the same plot. Although they appear to be different clumps, they all originate from the same plant and have basically the same growth. Therefore, similar materials can be selected for experimental screening based on the same plant.

[0049] Example:

[0050] I. Selection of Materials for Elephant Grass Cold Resistance Assessment

[0051] 1. Observation of the maturity of elephant grass seed stems in different regions

[0052] At the end of the growing season, the usable parts of the seed stems of different elephant grass varieties at different altitudes in Yunnan were observed. The results are shown in Table 1. As can be seen from the table, at the same growing time, the length of the usable seed stems decreased with increasing altitude, and the proportion of effective buds decreased (YM-1 is Yunmu No. 1, RY-4 is Reyan No. 4, DH is Dehong, GMY is Guimin Yin, and YM-2 is Yunmu No. 2).

[0053] Table 1

[0054]

[0055] 2. Observation of bud development at different parts of the stem node

[0056] At the end of the growing season, samples were taken from the stems of *Eriocheir sinensis* (2050m) for observation. The results showed that the upper and middle stem buds were weak or easily sprouted, with significant differences in sprouting status. The lower and middle stem buds were plump and mostly dormant. Figure 1 Comparison of multiple varieties revealed that bud development became more consistent from the third to the last yellowed leaf downwards; buds close to the ground, due to their thicker scales, were also unsuitable for identification. Therefore, comparison of different bud states was used ( Figure 2 For laboratory identification, robust stem buds with plump buds and in a dormant state are preferred, and the stem nodes should be from the third to the last withered yellow leaf down to the 1st or 2nd node near the base.

[0057] 3. Field germination characteristics of seed stems at different locations and with different water loss times

[0058] Field observations showed a strong correlation between stem development and leaf yellowing and maturity. To further verify the bud germination characteristics of different stem parts, bud germination experiments were conducted at different locations. The results showed that cuttings taken immediately after harvest could sprout from all nodes below the fully expanded leaves, but the germination rate decreased with prolonged storage. Five common Yunnan cultivars—RY-4, SM-1, DH, YM-1, JJC, and BD—were selected. Quantitative experiments were conducted on six stem segments (numbered 1-6 sequentially: ① third yellow leaf segment from the bottom, ② second yellow leaf segment from the bottom, ③ first yellow leaf segment from the bottom, ④ first green leaf segment, ⑤ second green leaf segment, ⑥ third green leaf segment) for different storage times. Three treatments were set up: 0 days, 5 days, and 10 days of storage (water loss time). The germination rates of different groups are shown in Table 2. The germination rates of RM-1 and RY-4 at 0, 5, and 10 days are shown in Table 2. Figure 3 , Figure 4 .

[0059] Table 2

[0060]

[0061] The above observations show that variety, different stem node positions, and different water loss times all have a certain impact on stem bud germination. For the same variety, the germination rate at different nodes is generally best near the boundary between withered yellow leaves and the first green leaf, showing a slight decreasing trend upwards. For the same variety at the same node, the germination rate generally decreases with prolonged water loss. From a laboratory identification perspective, to minimize differences in bud development and water loss, sampling from the stem nodes below the first yellow leaf to near the base, with a sampling time controlled within one day, yields the best results.

[0062] Based on the combined results of field surveys and experiments, the most suitable material for laboratory identification is the second-to-last yellow leaf down to the third node from the base. If material is insufficient, the third green leaf from the top can also be used.

[0063] II. Temperature and Time Selection for Elephant Grass Cold Hardiness Evaluation

[0064] 1. Research on the relationship between the duration of low temperature and frost damage

[0065] Using the relatively cold-hardy elephant grass variety "RY-4" as the research subject, the relationship between different durations of constant temperature at -2℃ and frost damage was studied. The experiment employed three replicates, with 30 stem nodes per replicate. After treatment, all materials were hydroponically cultured at 25℃ for 12 days, and the survival rates of roots and shoots were recorded. Detailed results can be found in [link to results]. Figure 5 .from Figure 5 As can be seen, the survival rates of both roots and shoots decreased with increasing freezing time. At the same time point, the root survival rate was higher than the shoot survival rate. Regression analysis showed that both exhibited a binomial regression relationship. Solving the regression formula, the times corresponding to 50% root and shoot survival rates were 81.5 h and 61.2 h, respectively, with an average of approximately 73 h. From a practical standpoint, 72 h (3 days) is more appropriate.

[0066] Using the cold hardiness index (root survival rate × 0.6 + bud survival rate × 0.4) as the evaluation index, and employing similar experimental methods as described above, the relationship between the weakly cold-resistant material RY-4 and the strongly cold-resistant material "6" (Table 3, No. 6) and frost damage under constant temperature conditions of -3℃ for different durations was studied. The experimental results are shown below. Figure 6 As shown in the figure, material "6" consistently outperformed material "RY-4" in cold resistance at all time points, consistent with field results. A binomial regression relationship was also observed between the two. Solving the regression formula, the times corresponding to 50% cold resistance index were 33.8 h and 5.7 h, respectively. This indicates that at a treatment temperature of -3℃, the time for cold-resistant materials to reach 50% mortality is extremely rapid, making it difficult to control in practice.

[0067] Based on the combined results of the -2℃ and -3℃ experiments, while the -3℃ test requires a shorter time, it is only suitable for materials with strong cold resistance. Although the -2℃ test takes longer, it is applicable to a wider range of materials; therefore, the -2℃ continuous 72-hour low-temperature stress scheme was adopted. Figure 7 (Illustrative diagram of the effects of hydroponics at -2℃ for 12 days on moderately cold-resistant materials (Table 3, No. 8) for different durations).

[0068] 2. Low-temperature germination research

[0069] Elephant grass is a C4 type high-light-efficiency plant, typically beginning to sprout and turn green when the temperature rises to around 15℃, with the optimal growth temperature above 25℃. The germination and growth characteristics at the low-temperature stage of around 15℃ are strongly correlated with the annual growth period and forage yield. This study selected eight materials with significant differences in field cold tolerance (material sources are shown in Table 3 below; field cold tolerance is graded from 1 to 5, with higher numbers indicating better cold tolerance) for early spring germination characteristics research. A 15℃ / 8℃ variable-temperature mode (a hydroponic mode with cyclical temperature changes of 15℃, 16h and 8℃, 8h) was set up, with a 20-25℃ constant-temperature hydroponic mode as a control. All treatments were cultured in an artificial incubator hydroponic system. After 10 days of cultivation, the germination characteristics of different materials were observed. Each treatment had three replicates, with 30 stem nodes per replicate. The main observation indicators included root sprouting rate, bud sprouting rate, shoot length, and root length. The average value of each treatment was calculated and then divided by the corresponding observation value at 25℃. The results are shown in Table 4. As shown in Table 4, a low-temperature environment of around 15℃ significantly inhibits the germination rate and growth rate of elephant grass compared to an environment of 20-25℃. The germination rate decreases by approximately 50%, while root development, shoot length, and root length decrease by over 80%. The trends are similar between the 15℃ constant temperature and 15 / 8℃ variable temperature models. Significant differences exist between materials; for example, material 6 performs better overall. From the perspective of the coefficient of variation, all coefficients of variation for the 15 / 8℃ variable temperature model are greater than those for the 15℃ constant temperature model, indicating that the 15 / 8℃ variable temperature model more fully reflects individual differences and is therefore more suitable for laboratory identification standards.

[0070] Table 3

[0071]

[0072] Table 4

[0073]

[0074] 3. Correlation analysis between low-temperature germination and cold resistance

[0075] The study employed a combination of low-temperature germination at 15 / 8℃ and low-temperature stress at -2℃ for 72 hours (4 hours at 4±0.5℃, followed by 72 hours at -2℃, and then 6-8 hours at 4±0.5℃, with the entire process conducted in a dry state). Twenty-five elephant grass resource numbers were collected, and a correlation analysis was performed between low-temperature germination and cold tolerance. The results (Table 5) show a certain correlation between low-temperature germination and laboratory cold tolerance evaluation results (R0). 2 =0.69~0.73). That is, the rapid germination at low temperatures reflects the overall cold resistance of the material to some extent. However, further individual analysis results show that there is no significant correlation between low-temperature germination inside the material and survival of frost damage.

[0076] Table 5

[0077]

[0078] 4. Selection scheme for relevant parameters

[0079] (1) The optimal stress scheme is -2℃ for 72 hours. This scheme is suitable for the cold resistance evaluation of most elephant grass materials. At the same time, it has low requirements for the experimental site. Generally, cold storage for fruit and vegetable preservation can meet the site requirements, and it is easy to achieve large-scale processing.

[0080] (2) Cold resistance indicators can be divided into two indicators: low-temperature stress survival rate and recovery rate. Actual observation results show that roots are more cold-resistant than buds, and the number of root points is greater, so external damage has less impact on the results than on buds. Therefore, when combining the survival results of roots and buds to represent the survival rate, roots and buds should be given different weights. In addition, in order to eliminate experimental errors caused by non-experimental factors, using indicators under non-stress conditions for correction can further ensure the objectivity of the results. Correlation analysis results show that low-temperature germination is strongly correlated with cold resistance. Based on the above reasons, the calculation formulas for relevant indicators in this scheme are as follows:

[0081] Cold hardiness index = low temperature germination rate × 0.3 + stress survival rate × 0.3 + recovery rate × 0.4 stress root point survival rate stress bud survival rate;

[0082] Survival (germination) rate = Survival rate of stressed root points / Survival rate of non-stressed root points × 0.6 + Survival rate of stressed buds / Survival rate of non-stressed buds × 0.4;

[0083] Recovery rate = (Struck root length / Non-stressed root length × 0.4) + (Struck seedling height / Non-stressed seedling height × 0.3) + (Struck leaf number / Non-stressed leaf number × 0.3)

[0084] III. Application

[0085] 1. Test Methods

[0086] Sixty elephant grass germplasm resources were collected in early winter and subjected to laboratory evaluation based on three indicators: low-temperature rapid germination, cold resistance survival rate, and recovery ability.

[0087] The experimental steps are as follows:

[0088] (1) When the first frost arrives, collect 60 elephant grass germplasm resources from the elephant grass resource nursery (each germplasm resource material is collected as a single plant clump). Collect the seed stems from the yellow leaves down to the third node from the bottom. Collect 200 elephant grass nodes containing a single dormant single bud from each germplasm resource material (the elephant grass node cut is 45°, 3cm long below the node and 5cm long above the node).

[0089] (2) Each germplasm resource material was divided into 4 parts, each with 50 nodes; Part 1 was placed in an artificial climate chamber and hydroponically cultured for 10 days under the variable temperature mode of 15℃ / 16h and 8℃ / 8h; Part 2 was placed in hydroponically cultured at 20-25℃ for 10 days as the control of Part 1; Part 3 was placed in constant temperature drying treatment at -2℃ / 72h for 72h, and then transferred to hydroponics at 20-25℃ for 10 days; Part 4 was placed in room temperature drying treatment for 72h, and then transferred to hydroponics at 20-25℃ for 10 days as the control of Part 3.

[0090] (3) After hydroponics, the root point germination rate, average root length, seedling survival rate, seedling height, number of leaves and other indicators of the four materials with the same number were counted respectively, and the low temperature germination rate, stress survival rate and recovery rate were calculated.

[0091] The cold hardiness index (calculation index of cold hardiness × 0.3 + survival rate under stress × 0.3 + recovery rate × 0.4) was calculated for different materials using three indicators: low-temperature germination rate, survival rate under stress, and recovery rate. Using a cold hardiness index ≥ 0.7 as the standard, seven potentially highly cold-hardy materials were finally screened and numbered YX2401, YX2402, YX2403, YX2404, YX2405, YX2406, and YX2407.

[0092] Using the weakly cold-resistant material RY-4 as a control, seven selected materials were transplanted to an altitude of 2000m after low-temperature stress (-2℃ / 72h treatment). They were planted individually at a spacing of 60cm × 60cm, with different materials planted alternately. The control RY-4 consisted of non-stressed hydroponic seedlings grown at the same time. Field management measures were the same. The tolerance of the stem tip at the same node (6th node from the base upwards) to the first frost of the planting year was observed. The results showed that the overall frost damage to the stem tips of the laboratory-selected strongly cold-resistant materials in late autumn and early winter was significantly less than that of the control RY-4. Figure 8 Left 1, the rest from left to right are YX2401-YX2407), among which YX2402, YX2405 and YX2406 are almost undamaged. After overwintering and turning green in the second year ( Figure 10 The observations included the greening time, rootstock rate (number of greening clumps / number of clumps before overwintering and rootstock rate (greening seedlings / number of stubble), and greening seedling height. The results (Table 6 below) showed that the strong cold-resistant materials screened in the laboratory greened up 5-10 days earlier the following year, with a rootstock rate increase of 37.3-47.1% and a plant growth rate increase of 81.6-137.5%. That is, the strong cold-resistant materials had a significantly improved overwintering and greening ability compared with the control weak cold-resistant materials. Using a rootstock rate ≥80% and a plant growth rate ≥180% as the standard, the 7 materials were screened again, and finally, 3 materials, YX2402, YX2405, and YX2406, were selected as candidate new lines for variety comparison trials at an altitude of 2000m.

[0093] Table 6

[0094]

[0095] 2. Laboratory screening of naturally overwintered stems

[0096] In years with unusual climate change (warm winters), a small number of shoots of elephant grass can survive the winter at an altitude of 2000m. These shoots may contain abundant cold-hardiness resources. Using this method, a large-scale laboratory screening was conducted on overwintered shoots collected from Xiaoshao, Kunming (1960m) in the RY-4 planting area.

[0097] The main steps differ somewhat from those of the aforementioned seven highly cold-resistant materials. The specific steps are as follows:

[0098] (1) Collect overwintering robust stems and buds of the same variety (RY-4) from individual plant clumps in the field, and cut them into single bud segments (cut at 45°, 3cm below the node and 5cm above the node).

[0099] (2) Randomly select single buds from 3 clumps and mix them. Divide them into 3 portions with 50 single buds in each portion, using the same method as described above. Place the 3 portions of material in an artificial climate chamber with variable temperature mode of 15℃ / 16h and 8℃ / 8h for 10 days of hydroponics, and place them in an indoor temperature of 20-25℃ for 0h and 72h for 10 days of hydroponics. The relevant results are used as control data for calculating the low-temperature rapid sprouting, cold resistance survival rate and recovery of all clumps of the same variety.

[0100] (3) Place the remaining single bud segments in a cold storage at -2℃ / 72h for 72h and then transfer them to a hydroponic system at 20-25℃ for 10 days.

[0101] After the treatment, observations revealed significant differences in survival rate and recovery growth rate among different RY-4 clumps after low-temperature stress treatment. Figure 11 The study used a weighted analysis of three laboratory indicators for single-plant clump germination at low temperatures, survival rate under stress, and recovery rate (low-temperature germination rate × 0.3 + survival rate under stress × 0.3 + recovery growth rate × 0.4). All clumps of the same variety with a cold hardiness index ≥ 0.7 were grouped into a single strongly cold-hardy material, designated YX25-48. Following the experimental procedures described above, and using greenhouse seedlings of the same variety as a control, field evaluation was conducted at an altitude of 2000m. Field evaluation results at the first frost indicated that YX25-48 showed stronger frost resistance in its stems and buds. Figure 9 Left 1, left 2, and left 3 show the longitudinal section and surface morphology of RY-4 stem bud frost damage, respectively; the greening process was significantly accelerated ( Figure 12 After turning green again, its growth rate is also faster. Figure 13 ).

[0102] The aforementioned application cases demonstrate that the combined laboratory and field approach of this scheme is reliable. Compared to traditional direct field screening, this scheme shortens the time from material screening to the determination of candidate new lines by at least one year, and reduces the scale of field trials by more than 80%. Field evaluation indicators should separately include three metrics: greening time, rootstock rate, and plant emergence rate. Overwintering ability is calculated based on the rootstock rate and plant emergence rate.

[0103] The criteria or calculation formulas for each indicator are as follows:

[0104] (1) Greening time: Based on overall dynamic observation, the greening time is determined by the fact that more than 30% of the clumps have more than one newly sprouted seedling;

[0105] (2) Sprouting rate: 30 days after the greening period, 20 clumps were observed diagonally along the plot. The number of new shoots (new shoots in the soil outside the stubble) ≥1 was considered the number of sprouts. Sprouting rate = number of sprouts / total number of plants.

[0106] (3) Sprouting rate: Survey was conducted 30 days after the greening period, with 20 plant clumps observed diagonally along the plot. The number of stubble and all new seedlings ≥5cm were counted for each plant clump. Sprouting rate = number of seedlings / number of stubble.

[0107] (4) Cold resistance overwintering ability = rootstock rate × 0.4 + plant growth rate × 0.6.

[0108] The above embodiments are only used to illustrate the technical solutions of the present invention, and are not intended to limit it. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for identifying and screening the cold resistance of elephant grass, characterized in that, The method includes the following steps: S0. Several elephant grass materials to be identified were asexually propagated to form a certain number of populations to meet the experimental sampling requirements; S1. Take a stem segment containing a single dormant bud from the second to last yellow leaf to the third node from the base of an elephant grass plant that was asexually propagated before overwintering as the identification material. S2. The above-mentioned identification materials were evenly divided into Experimental Group 1, Control Group 1, Experimental Group 2, and Control Group 2. The identification materials of the experimental group were hydroponically cultured for 7-14 days under a variable temperature mode of 15℃, 16h and 8℃, 8h; the identification materials of the control group were hydroponically cultured for 7-14 days under a constant temperature mode of 20-25℃. The identification materials of Experimental Group 2 were placed at a low temperature of 4±0.5℃ for 4 hours, then transferred to a constant temperature of -1℃ to -3℃ for 60-84 hours, then placed at a low temperature of 4±0.5℃ for 6-8 hours, and then hydroponically treated at room temperature of 20-25℃ for 7-14 days; the identification materials of Control Group 2 were placed at a constant temperature of 20-25℃ for the same time as Experimental Group 2, and then hydroponically treated at room temperature of 20-25℃ for 7-14 days. S3. Record the root germination rate of experimental group 1 as the low-temperature root germination rate, and the root germination rate of control group 1 as the control root germination rate; the average shoot length of experimental group 1 is the low-temperature average shoot length, and the average shoot length of control group 1 is the control average shoot length; calculate the root sprouting ability, bud sprouting ability, and low-temperature germination ability of experimental group 1, where root sprouting ability = low-temperature root germination rate / control root germination rate; bud sprouting ability = low-temperature average shoot length / control average shoot length; low-temperature germination ability = root sprouting ability × 0.6 + bud sprouting ability × 0.

4. S4. Record the root germination rate, average root length, seedling survival rate, seedling height, and number of leaves for experimental group 2 and control group 2; calculate the stress survival rate and recovery rate of experimental group 2, where the stress survival rate = (root germination rate of experimental group 2 / root germination rate of control group 2) × 0.6 + (seedling survival rate of experimental group 2 / seedling survival rate of control group 2) × 0.4; recovery rate = ((average root length of experimental group 2 / average root length of control group 2) + (seedling height of experimental group 2 / seedling height of control group 2) + (number of leaves of experimental group 2 / number of leaves of control group 2)) ÷ 3; S5. Calculate the cold resistance index of the identified material based on the above-mentioned low-temperature germination rate, stress survival rate and recovery rate, wherein the cold resistance index = (low-temperature germination rate × 0.3 + stress survival rate × 0.3 + recovery rate × 0.4).

2. The method according to claim 1, characterized in that: In step S1, the cut of the material being identified is at a 45° angle, with a length of 3 cm below the segment and 5 cm above the segment.

3. The method according to claim 1, characterized in that: In step S1, when there is insufficient identification material obtained from the same plant of elephant grass, the material is taken from the third green leaf upwards.

4. The method according to claim 1, characterized in that: In step S2, the identification materials of both experimental group 2 and control group 2 were subjected to constant temperature treatment in a dry state.

5. The application of the method as described in any one of claims 1-4 in the cultivation of new cold-resistant elephant grass varieties.

6. The application according to claim 5, characterized in that: The application method includes the following steps: (1) Elephant grass seedlings with a cold hardiness index of 0.7 or above were temporarily planted in a greenhouse and transplanted to an altitude of 2000m in mid-to-late March. The rooting rate and plant growth rate were counted 30 days after the greening up the following year, and the cold hardiness overwintering ability was calculated. The cold hardiness overwintering ability = rooting rate × 0.4 + plant growth rate × 0.6; (2) Select surviving materials with cold resistance to winter ≥ 0.8 to establish clones as materials for subsequent new line breeding.

7. The application according to claim 6, characterized in that: The greening time is defined as the period during which at least 30% of the plant clumps have at least one new sprout.

8. The application according to claim 6, characterized in that: The germination rate is calculated as: number of germination sites / total number of sites; the plant emergence rate is calculated as: number of seedlings / number of stubble.

Images