Plasma mutation breeding method for callus of hybrid pennisetum alopecuroides' Bangdel No.1 '

Abstract

The invention discloses a plasma mutation breeding method for callus of hybrid pennisetum alopecuroides' Bangdel No.1 '. According to the method, the callus induced by the Bangdel No.1 seed is used as a mutagenesis base material, an ARTP radiation mutagenesis technology is combined with plant tissue culture, and the mutagenesis effect of ARTP radiation on the hybrid pennisetum callus is explored from different aspects of phenotype, physiology and molecules in different stages of callus growth and plant regeneration. Furthermore, a low-temperature stress test is carried out on the regenerated plant, the morphological and physiological index differences of a mutagenesis plant and a control plant before and after low-temperature stress treatment are observed, mutagenesis strains with cold-resistant potential in the present generation are screened, and the cold-resistant hybrid pennisetum germplasm is obtained. The invention provides theoretical and material basis for the breeding of new germplasm of cold-resistant hybrid pennisetum alopecuroides.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology breeding, and in particular relates to a plasma mutagenesis breeding method for 'Bonde No. 1' hybrid Pennisetum callus. Background Art

[0002] Hybrid Pennisetum (Pennisetum glaucum × P. purpureum) is an F1 interspecific triploid hybrid developed from Pennisetum glaucum L. (female) and Pennisetum purpureum Schumach. (male). It boasts high yield and excellent quality, making it a staple grass species in the vast areas south of the Yangtze River in my country. Hybrid Pennisetum is rich in various amino acids and, when used as a feed, can significantly increase meat and milk production and quality. Its high lignocellulose content also allows for the production of cellulosic ethanol and biogas fermentation, making it considered one of the most promising new energy plants for the future.

[0003] Hybrid Pennisetum has high yield and rich nutritional value, and is considered one of the most promising forage and energy plants. However, its production is greatly affected by temperature. Improving the cold resistance of Pennisetum will help expand its cultivation area. The effective mutation frequency produced by traditional breeding methods is low, and the breeding period is also long. Mutation breeding technology uses physical and chemical factors to induce genetic variation in crops, forming genotypes that are difficult to obtain by traditional breeding, and further screening plants with target traits. This breeding method combines conventional breeding with modern physical and chemical technologies, greatly shortening the breeding time, and can provide rich original materials for the breeding of new varieties. Common sources of physical radiation mutagenesis include 60 Co-γ rays, ultraviolet rays, X-rays, and EMS are commonly used chemical mutagens. Physical and chemical mutagenesis can cause a variety of mutations, including point mutations and chromosomal aberrations. The distribution and properties of these mutations across the genome are non-directional.

[0004] Cold plasma is an ionized gas composed of photons, electrons, molecules, and excited atoms. During mutagenesis, collisions between electrons, atoms, and molecules produce a rich array of reactive species. Different reactive species interact with DNA at different sites, resulting in varying preferences for genetic mutations. This results in a wide range of induced mutations and a high mutation rate. Compared to traditional physical mutagenesis methods, cold plasma can generate more genes and is safer, leading to its increasing popularity in recent years. A Tsinghua University team, incorporating the fundamental principles of cold plasma, has developed a novel atmospheric pressure radio frequency glow discharge cold plasma generator. This uses radio frequency glow discharge of high-purity helium at atmospheric pressure to generate a high-energy cold plasma at temperatures between 25°C and 40°C. This cold plasma has been named atmospheric and room temperature plasma (ARTP). Under atmospheric pressure, ARTP can induce mutations in various organisms at room temperature. Within an appropriate electrode spacing, the plasma generator can discharge in alpha and gamma modes, generating a large number of active particles that can penetrate cells and effectively induce mutations. In addition, ARTP's operating conditions are relatively mild, the treatment process is safe and radiation-free, and the gene damage intensity is high. Currently, a mature technical system has been established in the field of microbial breeding and has been applied industrially. However, research in the field of plant genetic improvement is still in the exploratory stage. Compared with microbial systems, plant cells have a more complex response mechanism to ARTP mutation due to their unique cell wall structure, genomic complexity, and tissue heterogeneity. Currently, there are two main ways to apply ARTP mutation in plants. The first is to directly treat different plant tissue materials with ARTP mutations. The second is to use ARTP compound preparations to mix plant seeds or directly spray them to achieve a yield increase effect. Summary of the Invention

[0005] One object of the present invention is to provide a plasma mutagenesis method for 'Bangde No. 1' hybrid Pennisetum callus.

[0006] The plasma mutagenesis method of 'Bangde No. 1' hybrid Pennisetum callus provided by the present invention comprises the following steps: subjecting 'Bangde No. 1' hybrid Pennisetum callus to ARTP radiation mutagenesis; the ARTP radiation mutagenesis time can be 10-20 minutes, specifically 10 minutes or 20 minutes, preferably 20 minutes.

[0007] In the plasma mutagenesis method for 'Bonde No. 1' hybrid Pennisetum callus, the ARTP radiation mutagenesis conditions are as follows: power 360W, air flow 15SLM, rotation speed 10r / s, and the distance between the highest point of each sample plate and the injection port is 2mm.

[0008] In the plasma mutagenesis method for the callus tissue of 'Bonde No. 1' hybrid Pennisetum, an ARTP breeding apparatus is used for ARTP radiation mutagenesis.

[0009] In the plasma mutagenesis method of the 'Bangde No. 1' hybrid Pennisetum callus, the 'Bangde No. 1' hybrid Pennisetum callus is a mature embryonic callus with a size of 5×5 mm.

[0010] In the plasma mutagenesis method for 'Bonde No. 1' hybrid Pennisetum callus, the method further comprises the step of inducing and culturing 'Bonde No. 1' hybrid Pennisetum seeds to obtain 'Bonde No. 1' hybrid Pennisetum callus before the ARTP radiation mutagenesis.

[0011] Furthermore, the method of "inducing culture of 'Bonde No. 1' hybrid Pennisetum seeds to obtain 'Bonde No. 1' hybrid Pennisetum callus tissue" may include the following steps: disinfecting 'Bonde No. 1' hybrid Pennisetum seeds to obtain disinfected seeds, inducing culture of the disinfected seeds in an induction culture medium to obtain 'Bonde No. 1' hybrid Pennisetum callus tissue.

[0012] Furthermore, the disinfection method includes the following steps: taking full and mature 'Bonde No. 1' Pennisetum seeds, rinsing them with running water for 2 hours, placing them in a clean bench, soaking them in 75% alcohol for 30 seconds, washing them with sterile water 2-5 times, shaking them with a mixed solution of 4% sodium hypochlorite solution + Tween 20 for 15 minutes, and washing them with sterile deionized water until no foam is generated.

[0013] The induction culture conditions may be: performing induction culture at 25±2° C. in the dark for 2-4 weeks.

[0014] The solvent of the induction culture medium is water, and the solvents and their concentrations are as follows: MS 4.43 g / L, maltose 30 g / L, 2,4-D 5 mg / L, 6-BA 1 mg / L, proline 1 g / L, NAA 0.8 mg / L, and agar 8 g / L.

[0015] The pH of the induction medium is 5.83-5.85.

[0016] In some embodiments, the induction culture further includes a subculture step.

[0017] The subculture conditions are as follows: culture at 25±2° C. in the dark, and replace the subculture medium every two weeks.

[0018] The solvent of the culture medium used in the subculture is water, and the solvents and their concentrations are as follows: MS 4.43 g / L, maltose 30 g / L, 2,4-D 5 mg / L, 6-BA 1 mg / L, proline 1 g / L, and agar 8 g / L.

[0019] The pH of the culture medium used in the subculture is 5.83-5.85.

[0020] Another object of the present invention is to provide a plasma-induced mutagenesis breeding method for 'Bangde No. 1' hybrid Pennisetum.

[0021] The plasma mutagenesis breeding method of 'Bangde No. 1' hybrid Pennisetum provided by the present invention comprises the following steps: performing ARTP radiation mutagenesis on 'Bangde No. 1' hybrid Pennisetum callus according to the plasma mutagenesis method of 'Bangde No. 1' hybrid Pennisetum callus to achieve mutagenesis breeding.

[0022] In the plasma mutagenesis breeding method for the 'Bonde No. 1' hybrid Pennisetum, the ARTP radiation mutagenesis further includes a step of screening cold-resistant plants.

[0023] The plasma-induced mutagenesis breeding method for the 'Bonde No. 1' hybrid Pennisetum may comprise the following steps:

[0024] 1) ARTP radiation mutagenesis was performed on the callus of 'Bangde No. 1' hybrid Pennisetum to obtain the mutagenized callus;

[0025] 2) performing differentiation culture on the induced callus to obtain differentiated budding tissue;

[0026] 3) performing rooting culture on the differentiated sprouted tissue to obtain rooted seedlings;

[0027] 4) transplanting the rooted seedlings into soil for cultivation to obtain regenerated seedlings;

[0028] 5) subjecting the regenerated seedlings to low temperature treatment to obtain cold-resistant plants through screening.

[0029] In step 2), the differentiation culture conditions were 25±2°C, 16h light / 8h dark, and a light intensity of 200 μmol / m 2 / s.

[0030] The solvent of the differentiation culture medium used in the differentiation culture is water, and the solvents and their concentrations are as follows: MS 4.43 g / L, sucrose 30 g / L, NAA 1 mg / L, 6-BA 3 mg / L, and agar 8 g / L.

[0031] In step 3), the rooting culture conditions are 25±2°C, 16h light / 8h dark, and a light intensity of 200 μmol / m 2 / s.

[0032] The solvent of the rooting medium used in the rooting culture is water, and the solvent and its concentration are as follows: MS 2.215 g / L, maltose 30 g / L, and agar 8 g / L.

[0033] In the step 4), the root length of the rooted seedlings is 1 cm.

[0034] The culture conditions were 25±2°C, 16 h light / 8 h dark, and a light intensity of 200 μmol / m 2 / s.

[0035] The culturing time is at least one month.

[0036] The soil is a mixed seedling medium of vermiculite and coconut soil. During the transplanting, the root system of the seedlings is protected to prevent root damage and seedling death. The seedlings are covered and moisturized for the first 7 days after transplanting.

[0037] In the step 5), the low-temperature treatment is carried out at 0° C. for 1 day.

[0038] In the step 5), the cold-resistant plants are screened according to the semi-lethal temperature and / or membership function analysis method.

[0039] The method of using semi-lethal temperature to screen cold-resistant plants is a method well known to those skilled in the art, and specifically comprises the following steps: collecting leaves from the same leaf position of the plant after low temperature treatment, rinsing them clean and drying the surface moisture. 0.5 g of leaves were collected from each sample using a hole punch, and 5-7 samples were collected from each plant. They were placed in a low temperature cycler and treated at 2°C, 0°C, -2°C, -4°C, -6°C and -8°C, with each temperature gradient treated for 2 hours. The leaves after low temperature stress were immersed in a test tube containing 30 mL of deionized water, shaken for 2-3 hours, and then allowed to stand for 20-30 minutes to measure the initial conductivity S1. The leaves were treated in a boiling water bath for 10 minutes, and the boiling conductivity S2 of the leaf samples was measured after cooling to room temperature. Electrolyte permeation rate = S1 / S2×100%. The logistic regression equation for fitting the electrolyte permeation rate is: y = K / (1+ae -bx y is the electrolyte exudation rate of the leaf, x is the treatment temperature, K is the limiting electrolyte exudation rate, and a and b are equation parameters. After fitting the equation to determine the half-lethal temperature, the low-temperature-treated plants were ranked in descending order of their half-lethal temperatures. Plants with the highest rankings were considered cold-resistant.

[0040] The method of screening cold-resistant plants using the membership function analysis method is a method well known to those skilled in the art, and specifically comprises the following steps: detecting the phenotypes of the plants after low-temperature treatment (including plant height, number of tillers, leaf length, leaf width, main stem diameter, internode length), osmotic regulation substance indicators (including malondialdehyde, proline, soluble sugar, relative conductivity), photosynthetic pigment indicators (including total chlorophyll), antioxidant enzyme activities (including CAT, POD, SOD) and relative expression levels of cold-resistant genes (including PgCAT, PgCLH, PgNHX, PgP5CS), and then performing principal component analysis based on the test results of each indicator to screen out the main component factors, and then calculating the principal component factor scores and comprehensive scores (D values) of the plants after each low-temperature treatment, and finally sorting the plants after low-temperature treatment in descending order according to the D values, and the plants with the highest ranking are cold-resistant plants.

[0041] In some embodiments, cold-resistant plants can be screened based on the semi-lethal temperature and membership function analysis method, which specifically includes the following steps: using the semi-lethal temperature and membership function analysis method to rank the cold resistance of plants after low-temperature treatment, respectively, and screening the top-ranked plants (such as the top ten plants) using the two methods respectively. Plants screened out by both methods are cold-resistant plants.

[0042] The last object of the present invention is to provide any one of the following applications a1) to a10):

[0043] a1) Use of any of the above methods in promoting callus differentiation of Pennisetum hybridum;

[0044] a2) application of any of the above methods in promoting rooting of hybrid Pennisetum callus;

[0045] a3) application of any of the above methods in promoting seedling formation of hybrid Pennisetum callus;

[0046] a4) application of any of the above methods to increase the number of hybrid Pennisetum plants;

[0047] a5) Use of any of the above methods to increase the tillering number of hybrid Pennisetum;

[0048] a6) Use of any of the above methods for increasing the leaf size of hybrid Pennisetum;

[0049] a7) Use of any of the above methods in regulating the content of osmotic regulating substances in hybrid Pennisetum;

[0050] a8) Use of any of the above methods in regulating the antioxidant enzyme activity of hybrid Pennisetum;

[0051] a9) Application of any of the above methods in improving the cold resistance of hybrid Pennisetum;

[0052] a10) Use of any of the above methods in preparing new cold-resistant hybrid Pennisetum germplasm.

[0053] Any of the above-mentioned increasing the size of hybrid Pennisetum leaves is embodied by increasing the length and / or width of the hybrid Pennisetum leaves.

[0054] Any of the above-mentioned regulation of the content of osmotic regulating substances in hybrid Pennisetum is embodied in reducing the malondialdehyde content in hybrid Pennisetum and / or increasing the soluble sugar content in hybrid Pennisetum.

[0055] Any of the above-mentioned regulation of the antioxidant enzyme activity of hybrid Pennisetum is reflected in increasing the CAT enzyme activity of hybrid Pennisetum.

[0056] This study uses callus induced from 'Bonde No. 1' hybrid Pennisetum seeds as the mutagenic substrate, combining ARTP radiation mutagenesis with plant tissue culture. The study investigates the phenotypic and physiological effects of ARTP on hybrid Pennisetum callus at different stages of callus growth and plant regeneration. Furthermore, by subjecting regenerated plants to low-temperature stress, differences in morphology, physiological, and molecular parameters between the induced and control plants before and after treatment were observed. This study screened strains with potential for cold tolerance during the induced phase, yielding cold-tolerant hybrid Pennisetum germplasm. This study provides a theoretical and material foundation for the selection and breeding of new cold-tolerant hybrid Pennisetum germplasm. BRIEF DESCRIPTION OF THE DRAWINGS

[0057] Figure 1 Figures AC and DF show the 'Bonde No. 1' hybrid Pennisetum callus after ARTP radiation mutagenesis. Figures AC and DF show the 10-minute ARTP treatment group, and Figures GI show the 20-minute ARTP treatment group.

[0058] Figure 2 Figure 1 shows the mutagenic effect of ARTP radiation on callus differentiation of 'Bangde No. 1'. Figures AC show the CK group at 10, 20, and 30 days of differentiation; Figures DF show the group treated with 10 min ARTP at 10, 20, and 30 days of differentiation; and Figures GI show the group treated with 20 min ARTP at 10, 20, and 30 days of differentiation.

[0059] Figure 3 This is the mutagenic effect of ARTP radiation treatment on the rooting of callus tissue of 'Bangde No. 1'.

[0060] Figure 4 The mutagenic effects of ARTP radiation on the plant height and tiller number of regenerated seedlings of 'Bangde No. 1'. Figures A and B show the plant height and tiller number of regenerated seedlings, respectively. *P < 0.05, **P < 0.01, and *P < 0.001. ns indicates no significant difference.

[0061] Figure 5 Figure 1 shows the mutagenic effect of ARTP radiation on the leaf length and width of regenerated seedlings of 'Bangde No. 1'. Figures A and B show the leaf length and width of regenerated seedlings, respectively. *P < 0.05, **P < 0.01, and *P < 0.001. ns indicates no significant difference.

[0062] Figure 6 Figure 1 shows the mutagenic effect of ARTP radiation on the main stem diameter and internode length of regenerated seedlings of 'Bangde No. 1'. Figures A and B show the main stem diameter and internode length of regenerated seedlings, respectively. *P < 0.05, **P < 0.01, and *P < 0.001. ns indicates no significant difference.

[0063] Figure 7 Figure 2 shows the effect of ARTP radiation mutagenesis on the MDA and soluble sugar contents of regenerated seedlings of 'Bangde No. 1'. Figures A and B show the MDA and soluble sugar contents of regenerated seedlings, respectively. *P < 0.05, **P < 0.01, and *P < 0.001. ns indicates no significant difference.

[0064] Figure 8 Figure 1 shows the mutagenic effect of ARTP radiation on the relative electrical conductivity and proline content of regenerated seedlings of 'Bangde No. 1'. Figures A and B show the relative electrical conductivity and proline content of regenerated seedlings, respectively. *P < 0.05, **P < 0.01, and *P < 0.001. ns indicates no significant difference.

[0065] Figure 9 Figure 1 shows the mutagenic effect of ARTP radiation on the chla and chlb contents in regenerated seedlings of 'Bangde No. 1'. Panels A and B show the chlorophyll a and b contents in regenerated seedlings, respectively. *P < 0.05, **P < 0.01, and *P < 0.001. ns indicates no significant difference.

[0066] Figure 10 Figure 1 shows the mutagenic effect of ARTP radiation on the total contents of car and chl in regenerated seedlings of 'Bangde No. 1'. Figures A and B show the carotenoid and total chlorophyll contents in regenerated seedlings, respectively. *P < 0.05, **P < 0.01, and *P < 0.001. ns indicates no significant difference.

[0067] Figure 11 Figure 1 shows the mutagenic effect of ARTP radiation on the antioxidant enzyme activities in regenerated seedlings of 'Bangde No. 1'. Figures A, B, and C show the CAT, POD, and SOD contents in regenerated seedlings, respectively. *P < 0.05, **P < 0.01, and *P < 0.001. ns indicates no significant difference.

[0068] Figure 12Figure 1 shows the mutagenic effects of ARTP radiation on the MDA and soluble sugar contents of regenerated seedlings of 'Bangde No. 1' after cold stress. Figures A and B show the MDA and soluble sugar contents, respectively. *P < 0.05, **P < 0.01, and *P < 0.001. ns indicates no significant difference.

[0069] Figure 13 Figure 1 shows the mutagenic effects of ARTP radiation on the proline content and relative conductivity of regenerated seedlings of 'Bangde No. 1' after cold stress. Figures A and B show the proline content and relative conductivity, respectively. *P < 0.05, **P < 0.01, and *P < 0.001. ns indicates no significant difference.

[0070] Figure 14 The results show the mutagenic effect of ARTP radiation on the chla and chlb contents in regenerated seedlings of 'Bangde No. 1' after cold stress. *P < 0.05, **P < 0.01, *P < 0.001, ns indicates no significant difference.

[0071] Figure 15 The results show the mutagenic effect of ARTP radiation on the total contents of car and chl in regenerated seedlings of 'Bangde No. 1' after cold stress. In the figure, *P<0.05, **P<0.01, *P<0.001, and ns indicates no significant difference.

[0072] Figure 16 Figure 1 shows the mutagenic effect of ARTP radiation on the antioxidant enzyme activities of regenerated seedlings of 'Bangde No. 1' after cold stress. Figures A to C show the CAT, POD, and SOD contents, respectively. *P < 0.05, **P < 0.01, and *P < 0.001. ns indicates no significant difference.

[0073] Figure 17 It is the relative expression level of cold-resistant genes in plants with cold-resistant potential under normal temperature and cold stress.

[0074] Figure 18 To determine the plant height of cold-resistant potential plants in the field.

[0075] Figure 19 To determine the number of tillers in the field of plants with cold-resistant potential.

[0076] Figure 20 The leaf length of plants with cold-resistance potential was measured in the field. *P<0.05 in the figure.

[0077] Figure 21 The leaf width of plants with cold-resistance potential was measured in the field. *P<0.05 in the figure.

[0078] Figure 22 The main stem diameter of plants with cold-resistance potential was measured in the field. *P<0.05 in the figure.

[0079] Figure 23 The internode length of plants with cold-resistance potential was measured in the field. *P<0.05 in the figure.

[0080] Figure 24 Figure 1 shows the mutagenic effect of ARTP radiation on the ADF content of cold-resistant plants of 'Bangde No. 1'. Figure A shows the ADF content of each ARTP radiation treatment group, and Figure B shows the ADF content of each individual plant. *P < 0.05 in the figures.

[0081] Figure 25 Figure 1 shows the mutagenic effect of ARTP radiation on the NDF content of cold-resistant plants of 'Bangde No. 1'. Figures A and B show the NDF content of each ARTP radiation treatment group and each individual plant, respectively. *P < 0.05.

[0082] Figure 26 Figure 1 shows the effect of ARTP radiation on the protein content of cold-resistant plants of 'Bangde No. 1'. Figure A shows the protein content of each ARTP radiation treatment group, and Figure B shows the protein content of each individual plant. *P < 0.05 in the figures.

[0083] Figure 27 Figure 1 shows the mutagenic effect of ARTP radiation on the chlorophyll content of cold-resistant plants of 'Bangde No. 1' before and after frost. Figure A shows the changes in total chlorophyll content in each radiation treatment group, and Figure B shows the changes in total chlorophyll content in each cold-resistant plant. *P < 0.05 in the figures. DETAILED DESCRIPTION

[0084] The present invention will be further described in detail below in conjunction with specific embodiments. The examples provided are only for illustrating the present invention and are not intended to limit the scope of the present invention. The examples provided below can serve as a guide for further improvements by those skilled in the art and are not intended to limit the present invention in any way.

[0085] Unless otherwise specified, the experimental methods in the following examples are conventional methods and were performed according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials and reagents used in the following examples, unless otherwise specified, were all commercially available.

[0086] The 'Bonde No. 1' hybrid Pennisetum seeds in the following examples are products of Zhengzhou Kaiyuan Grass Industry Company.

[0087] The MS in the following examples is a product of Phyto Technology, with the product number being M519.

[0088] The seedling culture medium in the following examples is a product of Melco.

[0089] Example 1: ARTP radiation-induced mutagenesis breeding method for 'Bonde No. 1' hybrid Pennisetum

[0090] 1. Experimental Methods

[0091] 1. Analysis of the mutagenic effect of ARTP radiation treatment on callus tissue

[0092] 1-1. Callus induction

[0093] (1) Take full and mature 'Bonde No. 1' Pennisetum seeds and rinse them with running water for 2 hours. Then put them into a clean bench, soak them in 75% alcohol for 30 seconds, wash them with sterile water 2-5 times, shake them with a mixed solution of 4% sodium hypochlorite solution + Tween 20 for 15 minutes, and wash them with sterile deionized water until no foam is produced. Place the seeds on sterile filter paper and let them dry on the surface.

[0094] (2) The sterilized seeds were placed in an induction medium and cultured in the dark at 25±2°C for 2-4 weeks. The solvent of the induction medium was water, and the solvents and their concentrations were as follows: MS 4.43 g / L, maltose 30 g / L, 2,4-D 5 mg / L, 6-BA 1 mg / L, proline 1 g / L, NAA 0.8 mg / L, agar 8 g / L, and the pH was adjusted to 5.83-5.85.

[0095] (3) After 4 weeks of induction culture, subculture was performed under the following conditions: incubation at 25 ± 2°C in the dark, with the subculture medium replaced every two weeks. The subculture medium was composed of water and the following solvents and concentrations: MS 4.43 g / L, maltose 30 g / L, 2,4-D 5 mg / L, 6-BA 1 mg / L, proline 1 g / L, agar 8 g / L, and the pH was adjusted to 5.83-5.85.

[0096] 1-2. ARTP mutagenesis treatment of callus tissue

[0097] (1) Mature embryonic callus of uniform state and approximately 5×5 mm in size was selected for mutagenesis.

[0098] (2) The ARTP breeding instrument was set at 360W power, 15 SLM airflow, and 10 rpm. The distance between the highest point of each plate sample and the nozzle was 2 mm. Three treatments were set: 0 min, 10 min, and 20 min, with three plates per treatment as replicates. The 0 min treatment group served as a control.

[0099] (3) The ARTP-induced callus and the control callus were transferred to differentiation medium at 25 ± 2 °C, 16 h light / 8 h dark, and a light intensity of 200 μmol / m 2The differentiation culture was carried out under the conditions of 1:1 / s. The solvent of the differentiation medium was water, and the solvents and their concentrations were as follows: MS 4.43 g / L, sucrose 30 g / L, NAA 1 mg / L, 6-BA 3 mg / L, and agar 8 g / L.

[0100] (4) After the callus tissue differentiated and sprouted, it was transferred to a rooting culture bottle containing rooting medium for rooting culture. The rooting culture conditions were 25±2℃, 16h light / 8h dark, and a light intensity of 200μmol / m 2 / s, and transplanted to the seedling medium after the root system grew to more than 1 cm. In the greenhouse (25±2℃, 16h light / 8h dark, light intensity of 200μmol / m 2 The rooting medium was composed of water, and the solvents and their concentrations were as follows: MS 2.215 g / L, maltose 30 g / L, and agar 8 g / L.

[0101] 1-3. Measurement indicators and methods

[0102] (1) Determination of callus differentiation rate

[0103] The callus tissue after mutagenesis was inoculated into differentiation medium, and photographed and counted every 10 days. The number of green regenerated buds of the callus tissue under each treatment dose was recorded, and the callus differentiation rate was calculated according to the following formula: Callus differentiation rate (%) = (number of callus tissues with green regenerated buds under the radiation time / total number of callus tissues under the radiation time) × 100%.

[0104] (2) Determination of callus rooting rate and seedling rate

[0105] The callus tissue transferred to the rooting medium was photographed and counted every 10 days, and the rooting rate was recorded.

[0106] After the roots grew to more than 1 cm, they were transplanted into the soil and the final seedling rate at each dose was calculated.

[0107] Rooting rate (%) = (number of calli that took root during the irradiation time / total number of calli during the irradiation time) × 100%.

[0108] Seedling rate (%) = (number of regenerated seedlings during the irradiation time / total number of callus tissues during the irradiation time) × 100%.

[0109] 2. Analysis of the mutagenic effect of ARTP irradiation on regenerated seedlings

[0110] The obtained regenerated seedlings were tested for phenotypic and physiological and biochemical indicators.

[0111] 2-1. Determination of phenotypic indicators of regenerated seedlings

[0112] The regenerated seedlings were tested for five morphological parameters: plant height, tiller number, leaf length and width, main stem diameter, and internode length. The phenotypic parameter testing methods are shown in Table 1.

[0113] Table 1. Phenotypic index determination methods

[0114]

[0115] 2-2. Determination of physiological and biochemical indicators of regenerated seedlings

[0116] (1) Relative conductivity

[0117] Select leaves from the same position on each plant, rinse thoroughly, dry, and cut into 1 cm segments. Weigh 5 g of leaves into a 50 mL centrifuge tube, add 20 mL of ultrapure water, and shake for 24 hours. Determine the initial conductivity (S1). Then, autoclave at 121°C for 15-20 minutes, cool, and shake for 24 hours. Determine the final conductivity (S2). Calculate relative conductivity (L) using the following formula: Relative conductivity (L) = S1 / S2.

[0118] (2) Soluble sugar, MDA, proline, CAT, POD, SOD

[0119] Soluble sugar, MDA, proline, CAT, POD, and SOD were all detected using kits from Beijing Solebaugh Company.

[0120] (3) Chlorophyll content

[0121] Weigh 0.5 g of leaves and soak them in 95% ethanol until the leaves turn pale. Measure the absorbance of the soaking solution at 665 nm, 649 nm, and 470 nm, and calculate the content according to the following relationship:

[0122] C a =13.95A 665 -6.88A 649 .

[0123] C b =24.96A 649 -7.32A 665 .

[0124] C x.c =(1000A 470 -2.05C a -114.8C b ) / 245.

[0125] C 总 =C a +C b .

[0126] Where: C a 、C b are the concentrations of chlorophyll a and b, respectively; C x.c is the total concentration of carotenoids; C 总 is the total chlorophyll concentration.

[0127] 3. Analysis of the mutagenic effect of ARTP radiation treatment on the cold resistance of regenerated seedlings

[0128] 3-1 Experimental Design

[0129] Regenerated seedlings from each ARTP-irradiated group were placed in an intelligent light incubator and treated at 0°C for 1 day. After the cold stress treatment, samples were taken for physiological index measurement or placed in a -80°C refrigerator for testing. Principal component analysis and membership function analysis were performed on the phenotype and various physiological indicators, and a comprehensive evaluation D value was calculated. The cold tolerance of the plants was evaluated by comparing the D values. The specific analysis method is as follows:

[0130] (1) Membership function value

[0131] Y(Xi)=(Xi-Xmin) / (Xmax-Xmin); where Xi represents the score of the i-th factor after principal component analysis, Xmin represents the minimum score of the i-th factor, and Xmax represents the maximum score of the i-th factor.

[0132] (2) Calculation of weights

[0133] Where Wi represents the importance of the i-th indicator; Pi represents the contribution rate of the i-th indicator.

[0134] (3) Comprehensive evaluation D value

[0135] Where D represents the comprehensive index value. The larger the D value is, the stronger its cold resistance is.

[0136] 3-2. Determination of physiological and biochemical indices of regenerated seedlings after cold stress

[0137] (1) Relative conductivity, soluble sugar, MDA, proline, CAT, POD, SOD, chlorophyll content

[0138] The determination methods of relative conductivity, soluble sugar, MDA, proline, CAT, POD, SOD and chlorophyll content are the same as above.

[0139] (2) Half-lethal temperature

[0140] Collect leaves from the same leaf position of each plant, rinse them clean and wipe off the surface moisture. Collect 0.5g of leaves for each sample with a hole punch, and collect 5-7 samples from each plant. Place them in a low-temperature cycler and treat them at 2℃, 0℃, -2℃, -4℃, -6℃ and -8℃, and treat them at each temperature gradient for 2h. Soak the leaves after low-temperature stress in a test tube containing 30mL deionized water, shake them for 2-3h, and then let them stand for 20-30min to measure the initial conductivity S1. Treat them in a boiling water bath for 10min, cool them to room temperature, and measure the boiling conductivity S2 of the leaf samples.

[0141] Electrolyte extravasation rate = S1 / S2×100%.

[0142] The logistic regression equation for electrolyte extravasation rate is: y = K / (1 + ae -bx ). Where y is the electrolyte permeation rate of the leaf, x is the treatment temperature, K is the limit electrolyte permeation rate, and a and b are equation parameters.

[0143] 3-3. Determination of expression levels of cold-resistance-related genes

[0144] (1) RNA extraction and cDNA synthesis

[0145] Plant total RNA was extracted using the Plant Total RNA Extraction Kit (Novozymes), following the kit instructions. First-strand cDNA was synthesized using the extracted total RNA as a template using reverse transcription according to the TaKaRa reverse transcription kit instructions. All samples were reverse transcribed to a uniform concentration of 1000 ng / μL.

[0146] (2) Primer design

[0147] Milletdb software (http: / / milletdb.novogene.com / home / ) was used to align the sequences of Pennisetum homologous genes with the ATCAT, ATCLH, PvP5CS, and PvNHX gene sequences published on NCBI. Specific primers for these Pennisetum homologous gene sequences were designed using IDT software. The internal reference gene was the actin 1 gene (PpACT1, NCBI: MT784734) from Elephant Grass. The primer sequences are shown in Table 2.

[0148] Table 2. Primer synthesis sequences

[0149]

[0150] (3) Real-time fluorescence quantitative PCR

[0151] Using cDNA as a template, quantitative PCR was performed using the primers listed in Table 2 according to the instructions of the TaKaRa Fluorescence Quantitation Kit on a QuanStudio 1 instrument. The internal reference gene was PgACT1. Two replicates were performed for each sample.

[0152] 4. Analysis of the mutagenic effects of ARTP radiation treatment on field indicators of cold-resistant potential plants

[0153] 4-1. Overview of the Experimental Site

[0154] The experimental site is the Jinan Grass Germplasm Resource Garden of Shandong Academy of Agricultural Sciences, located at 36°40'N, 116°57'E, with a temperate continental monsoon climate. July and August are the hottest months, with an average temperature of 26.4°C; December is the coldest month, with an average temperature of -0.2°C; the average annual temperature is 14.7°C, the average annual precipitation is 645 mm, the average annual sunshine duration is 2457.7 h, and the frost-free period is 178 days.

[0155] 4-2. Field phenotypic index determination

[0156] After two months of field growth, phenotypic indicators were measured on October 19 using the same method as above.

[0157] 4-3. Fiber content determination

[0158] Neutral Detergent Fiber (NDF) and Acid Detergent Fiber (ADF) were measured using an automatic cellulose analyzer (ANKOM A2000i). The specific steps are as follows:

[0159] (1) Sample preparation: Grind the sample to be tested through a 1 mm sieve and accurately weigh 0.5 g (W1) into a heat-resistant filter bag;

[0160] (2) Neutral washing treatment: add 100 mL of neutral detergent solution (containing 30 g / L sodium lauryl sulfate, 18.61 g / L disodium ethylenediaminetetraacetic acid, 6.81 g / L sodium tetraborate, and 4.56 g / L disodium hydrogen phosphate) and reflux at 100 °C for 1 h.

[0161] (3) Acidic washing treatment: The NDF residue was placed in 100 mL of an acidic detergent solution (20 g / L hexadecyltrimethylammonium bromide, 30 mL / L 98% sulfuric acid) and refluxed at 100° C. for 1 h.

[0162] (4) Washing and drying: Wash the residue with hot water and acetone in sequence, and dry it at 105°C to constant weight (W2).

[0163] NDF / ADF content (%) = W2 / W1×100%.

[0164] 4-4. Protein content determination

[0165] The nitrogen content was measured using an elemental analyzer (Rapid CS Cube; Elementar, Germany), and the protein content was calculated according to the following formula: protein content = 6.25 × nitrogen content.

[0166] 5. Data processing and analysis

[0167] The measured data were entered into Excel 2019, and the significance of differences in each measured indicator was calculated using IBM SPSS Statistics 26. Correlation, principal component analysis, and membership function analysis were performed on each indicator to determine the mutagenic effects of different ARTP treatments on the plants. Correlation heat maps were created using Origin, and histograms were constructed using GraphPad Prism. Each point represents a data point, and the distribution of scattered points reflects the degree of dispersion of each data set.

[0168] 2. Experimental Results

[0169] 1. Mutagenic effect of ARTP irradiation on callus tissue of 'Bonde No. 1'

[0170] (1) Mutagenic effects of ARTP irradiation on callus phenotypes of 'Bonde No. 1'

[0171] like Figure 1 As shown in the figure, after ARTP mutagenesis treatment, the texture and color of the hybrid Pennisetum 'Bonde No. 1' callus tissue underwent significant changes: with the increase of radiation time, the burnt yellow color of the callus surface deepened, the wrinkling phenomenon intensified, and the surface water content gradually decreased. After 20 minutes of ARTP treatment, some calli tissues showed obvious burnt brown, the epidermal tissue was dehydrated and had a leathery texture, and the degree of cell damage increased with the deepening of treatment dose.

[0172] (2) Mutagenic effect of ARTP irradiation on callus differentiation of 'Bonde No. 1'

[0173] like Figure 2 As shown, 10- and 20-minute ARTP treatments demonstrated high differentiation potential early in the differentiation process, with significantly higher differentiation rates than the CK group. The regenerated shoots were also less likely to wither. Between 10 and 30 days, the differentiation rate in each group increased significantly, stabilizing by day 40, reaching a maximum number of differentiated shoots (Table 3). In the differentiation medium, some differentiated shoots in the CK group often formed protrusions but failed to develop into complete shoots, or the differentiated shoots were generally small. However, ARTP radiation mutagenesis significantly promoted callus differentiation in 'Bangde No. 1,' accelerating callus differentiation and improving differentiation efficiency, resulting in a sufficient number of high-quality differentiated shoots.

[0174] Table 3. Mutagenic effect of ARTP treatment on callus differentiation of 'Bangde No. 1'

[0175]

[0176]

[0177] Note: Lowercase letters in the figure indicate significant differences (P<0.05).

[0178] (3) Mutagenic effect of ARTP irradiation on rooting of callus tissue of 'Bonde No. 1'

[0179] Pennisetum has a strong rooting ability and a well-developed root system. Except for some calli that have small regenerated buds or fail to take root due to high aging of the callus itself, or the buds turn yellow after taking root and cannot grow into regenerated plants, most of the calli that have differentiated into green buds in each treatment group can take root successfully. Figure 3 It can be seen that the rooting rate of the treatment group was slightly higher than that of the CK group (Table 4), and the regenerated seedlings in the 20-min ARTP treatment group had the largest root system, the highest number of tillers, and the plant stems were strong.

[0180] Table 4. Mutagenic effect of ARTP treatment on callus rooting of 'Bangde No. 1'

[0181] Radiation treatment time Rooting rate (%) CK (0min) <![CDATA[75.15±4.20 a ]]> 10min <![CDATA[77.59±2.51 a ]]> 20min <![CDATA[82.22±1.92 a ]]>

[0182] Note: Lowercase letters in the figure indicate significant differences (P<0.05).

[0183] (4) Mutagenic effects of ARTP irradiation on seedling formation of callus of 'Bangde No. 1'

[0184] The seedlings with healthy root systems were transplanted into a mixed seedling medium of vermiculite and coconut soil. During the transplanting process, special attention was paid to protecting the roots of the seedlings to prevent root damage and seedling death. During the first 7 days after transplanting, the seedlings were covered to keep them moist. Pennisetum prefers heat and humidity, so it should be watered more frequently during this period. As time passed, the differentiated buds gradually developed into plants, marking the successful completion of the process from callus regeneration to complete plants. In the end, 42 regenerated seedlings were obtained in the CK group, 52 regenerated seedlings were obtained in the 10min ARTP treatment group, and 30 regenerated seedlings were obtained in the 20min ARTP treatment group (the 20min treatment group lost part of the callus due to operational errors and was infected with bacteria, resulting in fewer seedlings than other groups).

[0185] 2. Mutagenic effect of ARTP irradiation on the phenotype of regenerated seedlings of 'Bangde No. 1'

[0186] Phenotypic observations were conducted on plants in each ARTP mutagenesis treatment group, and the results showed that the plants in the CK group showed obvious growth restriction characteristics, with plant height significantly lower than that of the radiation treatment group, narrower leaves and more serious yellowing of leaves, significantly reduced number of new tillers at the base of the plants, and sparser group structure. Compared with the CK group, the plants in the 10min ARTP and 20min ARTP treatment groups showed significant growth advantages, with a significant increase in the diameter of the main stem, good development of the mechanical tissue of the stem, and significantly enhanced uprightness. In terms of leaf traits, the treatment group had obvious leaf biomass accumulation, and at the same time, the degree of leaf senescence was low, and the yellowing rate was significantly reduced. These morphological differences indicate that ARTP treatment at the appropriate time can effectively promote the morphological construction of Pennisetum. The specific results are as follows:

[0187] (1) Mutagenic effects of ARTP irradiation on plant height and tiller number of regenerated seedlings of 'Bangde No. 1'

[0188] Depend on Figure 4 A shows that there are differences in the effects of different ARTP radiation treatments on the plant height of 'Bonde No. 1' hybrid Pennisetum. The average plant height of the CK group is 49.12 cm. The 10-minute ARTP treatment has a significant promoting effect on the growth of the plant height of Pennisetum seedlings (P < 0.01), with the average plant height reaching 58.30 cm. When the treatment time is 20 minutes, the promoting effect on plant height is slightly lower than that of the 10-minute treatment, with an average plant height of 51.61 cm, which is not significantly different from the CK. Figure 4 B shows that there are differences in the effects of different ARTP radiation treatments on the tiller number of 'Bonde No. 1' hybrid Pennisetum. The 20-min ARTP treatment has a significant promoting effect on increasing the tiller number (P < 0.05), with an average tiller number of 3.1 per plant, which is significantly higher than that of the CK group. The average tiller number per plant in the 10-min ARTP mutagenesis treatment group is 1.76, which is slightly lower than that of the CK group, but there is no significant difference.

[0189] (2) Mutagenic effects of ARTP irradiation on leaf length and width of regenerated seedlings of 'Bangde No. 1'

[0190] Depend on Figure 5 A shows that the average leaf length of the CK group was 33.81 cm, and the average leaf lengths of the 10-min ARTP and 20-min ARTP treatment groups were 41.74 cm and 37.22 cm, respectively, which were both longer than those of the CK group. The 10-min ARTP treatment group had a significant promoting effect on plant leaf length (P < 0.001). Figure 5 As shown in Figure 2, the average leaf widths of the 10-min ARTP and 20-min ARTP treatment groups were 17.48 mm and 16.9 mm, respectively, which were significantly higher than those of the CK group (P < 0.0001).

[0191] (3) Mutagenic effects of ARTP irradiation on the main stem diameter and internode length of regenerated seedlings of 'Bangde No. 1'

[0192] Depend on Figure 6 A shows that the average main stem diameter of the CK group is 10.59 mm, and the average main stem diameters of the 10-min ARTP and 20-min ARTP treatment groups are 10.42 mm and 10.70 mm, respectively. There is no significant difference in the main stem diameters between the different treatment groups. Figure 6 As shown in Figure 2, the average internode lengths of the CK group and the 10-min ARTP treatment group were 5.71 cm and 5.802 cm, respectively, with no significant difference between the two groups. The average internode length of the 20-min ARTP treatment group was 6.36 cm, slightly higher than that of the CK group.

[0193] 3. Mutagenic effects of ARTP irradiation on physiological parameters of regenerated seedlings of 'Bangde No. 1'

[0194] (1) Mutagenic effects of ARTP irradiation on osmotic regulation substances in regenerated seedlings of 'Bangde No. 1'

[0195] Effects of ARTP radiation-induced mutagenesis on osmotic regulation substances in regenerated seedlings Figure 7 and Figure 8 As shown, the malondialdehyde content of regenerated seedlings decreased significantly with increasing irradiation time. The average malondialdehyde content in the CK group was 20.7369 nmol / g, while the average malondialdehyde content in the 10-min ARTP and 20-min ARTP treatment groups was 11.3978 nmol / g and 8.7342 nmol / g, respectively, representing a decrease of more than 50%. The soluble sugar content in the irradiated groups increased significantly, with the increase in the 20-min ARTP treatment group being significant (P < 0.01). The average soluble sugar content was 2.16 mg / g, 2.84 times that of the CK group. The relative conductivity of the 10-min ARTP and 20-min ARTP treatment groups was slightly lower than that of the CK group, with average decreases of 16.57% and 16.28%, respectively, but the difference was not significant. There was no significant difference in proline content between the CK and 20-min ARTP treatment groups. The proline content in the 10-min ARTP treatment group was slightly higher than that in the CK group, but the difference was not significant, with an average relative increase of 26.09%.

[0196] (2) Mutagenic effect of ARTP irradiation on the content of photosynthetic pigments in regenerated seedlings of 'Bangde No. 1'

[0197] Depend on Figure 9 and Figure 10It can be seen that ARTP radiation mutagenesis has little effect on the total chlorophyll and carotenoid contents of regenerated seedlings. The chlorophyll a content of the 10-min ARTP treatment group is higher, and its mean value is 1.04 times that of the CK group. The chlorophyll b content of the 10-min ARTP and 20-min ARTP treatment groups is slightly higher than that of the CK group, and their mean values are 1.31 and 1.63 times that of the CK group, respectively.

[0198] (3) Mutagenic effect of ARTP irradiation on antioxidant enzyme activity in regenerated seedlings of 'Bangde No. 1'

[0199] Depend on Figure 11 It can be seen that the CAT activity of the 10-min ARTP and 20-min ARTP treatment groups was higher than that of the CK group. The average CAT values of the 10-min ARTP and 20-min ARTP treatment groups were 151.05 U / g and 145.73 U / g, respectively, and the increases were 75.58% and 69.39% relative to the CK group, respectively. There was no significant difference in POD activity among the groups. The average SOD activity of the 20-min ARTP treatment group was 156.32 U / g, which was 1.28 times that of the CK group.

[0200] 4. Analysis of the comprehensive effects of ARTP radiation on the phenotype and physiological indicators of regenerated seedlings at room temperature

[0201] A total of six principal components were extracted using principal component analysis (PCA), with a cumulative contribution rate of 72.421%, which can fully reflect the original data information. The first principal component mainly reflects plant growth characteristics, among which plant height (0.769), leaf length (0.724), and leaf width (0.603) have high loading values. The second principal component mainly characterizes the photosynthetic system and antioxidant capacity, with chlorophyll a (0.803), total chlorophyll (0.802), and carotenoids (0.773) showing significant positive loadings. The third principal component mainly reflects stress response characteristics, with proline (0.678) and catalase (0.614) having high positive loadings, while tiller number (-0.563) and internode length (-0.568) showed significant negative loadings. This suggests that plants may respond to stress conditions by increasing peroxidase activity and proline accumulation, but at the same time inhibiting tillering and internode elongation. The fourth principal component primarily reflects oxidative stress, with malondialdehyde (0.569) and relative conductivity (0.385) exhibiting positive loadings, while chlorophyll b (-0.621) showed a significant negative loading. The fifth principal component was primarily associated with osmotic regulation and antioxidant mechanisms, with superoxide dismutase (0.664), peroxidase (0.515), and relative conductivity (0.469) showing significant loadings. In the sixth principal component, catalase (0.380) and tiller number (0.400) exhibited positive loadings, while main stem diameter (-0.469) and peroxidase (-0.384) showed negative loadings (Table 5).

[0202] Table 6 shows the comprehensive evaluation results of different ARTP radiation treatment times on the indoor phenotype and physiological indicators of regenerated seedlings. The 20-min treatment group had the highest D value (0.441), ranking first, and had the most significant overall promoting effect on the regenerated seedlings, especially in factors such as the second principal component (0.644), the fifth principal component (0.500), and the sixth principal component (0.524). The 10-min treatment group (D=0.359) ranked second, and the CK group had the lowest D value (0.290). In summary, the 20-min ARTP radiation treatment had the best mutagenic effect on the growth and physiological state of the regenerated seedlings, while the untreated control group performed the worst, indicating that ARTP radiation treatment of appropriate time can effectively improve the characteristics of the regenerated seedlings.

[0203] Table 5. Principal component analysis of normal temperature phenotypes and physiological indices of ARTP irradiated regenerated seedlings

[0204]

[0205] Table 6. Comprehensive evaluation of indoor phenotypes and physiological indices of regenerated seedlings treated with ARTP radiation

[0206]

[0207] 5. Mutagenic effects of ARTP irradiation on regenerated seedlings of 'Bangde No. 1' after cold stress

[0208] (1) Mutagenic effect of ARTP irradiation on the content of osmotic regulatory substances in regenerated seedlings of 'Bangde No. 1' after cold stress

[0209] Effects of ARTP on the content of osmotic regulation substances in regenerated seedlings after cold stress Figure 12 and 13 As shown. Figure 12 A shows that the MDA content of different treatment groups increased significantly after cold stress (P < 0.01). The mean MDA content of the 10-min ARTP and 20-min ARTP treatment groups decreased by 26.78% and 19.1% respectively compared with the CK group, indicating that the cell membrane damage of the ARTP mutagenesis group was less than that of the CK group. Figure 12 B shows that cold stress can cause a significant increase in the soluble sugar content in plants (P < 0.05). The average soluble sugar content of each treatment group after cold stress increased by 186.8%, 95.89% and 60.47% respectively compared with the normal temperature treatment. The soluble sugar content of the ARTP treatment group 10 minutes after cold stress and the ARTP treatment group 20 minutes after cold stress increased by 60.91% and 58.57% respectively compared with the CK group, indicating that the ARTP mutagenesis group has a higher soluble sugar content than the CK group and has stronger cold resistance. Figure 13A shows that cold stress can cause a significant increase in proline content (P < 0.05). The average proline content of the 10-min ARTP mutagenesis group after cold stress is the highest, at 388.86 ug / g, an increase of 85.72% compared with CK. Figure 13 B shows that the relative conductivity of plants in each treatment group increased significantly after cold stress (P < 0.01). The mean relative conductivity of plants in each treatment group after cold stress were 76.43%, 71.21% and 68.65%, respectively. The relative conductivity of plants in the 10-min ARTP and 20-min ARTP treatment groups after cold stress decreased by 6.83% and 10.14%, respectively, compared with the CK group, indicating that cold stress can cause cell membrane damage and increase the chance of extravasation of contents. However, the relative conductivity of the ARTP treatment group was lower, the cell damage was less, and the cold resistance was stronger.

[0210] (2) Mutagenic effect of ARTP irradiation on the content of photosynthetic pigments in regenerated seedlings of 'Bangde No. 1' after cold stress

[0211] Effects of ARTP on photosynthetic pigment content in regenerated seedlings after cold stress Figure 14 As shown by Figure 14 A shows that the chlorophyll a content decreased significantly after cold stress. The mean chlorophyll a values of each treatment group after cold stress were 0.1874 mg / g, 0.2354 mg / g and 0.2352 mg / g, respectively. Figure 14 B shows that the chlorophyll b content decreased. After cold stress, the average chlorophyll b content of each treatment group was 0.0223 mg / g, 0.0272 mg / g and 0.0391 mg / g, respectively. The 10 min ARTP and 20 min ARTP treatment groups increased by 21.97% and 75.34% respectively compared with the CK group. Figure 15 A shows that the carotenoid content decreased after cold stress, and the carotenoid content in the 10-min ARTP treatment group decreased most significantly. After cold stress, the average carotenoid content of each treatment group was 0.0914 mg / g, 0.1059 mg / g, and 0.1178 mg / g, respectively. The carotenoid content in the ARTP treatment group increased by 15.86% and 28.88% compared with the CK group, respectively. Figure 15 B shows that the total chlorophyll content of each treatment group decreased after cold stress. The average total chlorophyll content of each treatment group after stress was 0.2075 mg / g, 0.2626 mg / g and 0.2743 mg / g, respectively. The total chlorophyll content of the 10 min ARTP and 20 min ARTP treatment groups increased by 26.55% and 32.19% compared with the CK group.

[0212] (3) Mutagenic effect of ARTP irradiation on antioxidant enzyme activities in regenerated seedlings of 'Bangde No. 1' after cold stress

[0213] Effects of ARTP on antioxidant enzyme activities in regenerated seedlings after cold stress Figure 16 As shown by Figure 16 A shows that CAT activity increased significantly after cold stress (P < 0.0001), and the CAT activity of the 10-min ARTP treatment group increased significantly. The mean CAT activity of the 10-min treatment group after cold stress was 3.61 times that of the CK group. Figure 16 B shows that POD activity increased significantly after freezing stress (P < 0.01). The mean POD activity of each treatment group after cold stress was 26080.6U / g, 34471.5U / g and 39552.5U / g, respectively. The POD activity of each treatment group after cold stress increased by 32.17% and 51.65% compared with the CK group. Figure 16 C shows that after cold stress, the SOD activity of each treatment group increased, with the mean values being 130.6U / g, 220.9U / g and 208.9U / g, respectively. After cold stress, the POD activity of each treatment group increased by 69.16% and 59.96% relative to that of the CK group, respectively.

[0214] 6. Screening and identification of plants with cold-resistant potential

[0215] After cold stress, the leaves of the plants showed obvious dehydration and dryness. After pruning and removing these damaged dead leaves, some plants showed regeneration ability, and the new tissues gradually turned green. Ultimately, 2 out of 42 plants in the CK group survived, with a survival rate of 4.76%. 6 out of 52 plants in the 10-minute ARTP treatment group survived, with a survival rate of 11.54%. 10 out of 30 plants in the 20-minute ARTP treatment group survived, with a survival rate of 33.33%. A preliminary screening of 17 plants with cold-resistant potential (1 plant in the CK group, 6 plants in the 10-minute ARTP treatment group, and 10 plants in the 20-minute ARTP treatment group) was conducted.

[0216] (1) Rapid screening of cold-resistant plants using semi-lethal temperature

[0217] The semi-lethal temperature (ST) can directly reflect a plant's cold tolerance. Further testing of the potential cold-tolerant plants was performed. Table 7 shows that the ST was -2.609°C for the CK group, -2.7346°C for the 10-min ARTP-treated group, and -3.497°C for the 20-min ARTP-treated group. With the exception of '20-24', '10-48', and '10-28', which had STs higher than those of the CK group, the STs of the remaining potential cold-tolerant plants were lower than those of the CK group, indicating improved cold tolerance. The STs of '10-36', '20-1', '20-18', '20-19', '20-23', and '20-25' were 130% lower than those of the CK group, indicating stronger cold tolerance.

[0218] Table 7. Mutagenic effects of ARTP irradiation on the semi-lethal temperature of regenerated seedlings of 'Bangde No. 1'

[0219]

[0220]

[0221] (2) Membership function analysis method to screen cold-resistant potential plants

[0222] (2-1) Determination of relative expression levels of cold-resistant genes in plants with cold-resistant potential

[0223] Determination of the relative expression of cold-resistant genes in plants with cold-resistant potential can better analyze their cold-resistant ability at the molecular level. Figure 17 As shown, PgCAT expression levels in the ARTP-treated group were higher than in the CK group at room temperature. In the irradiated group, the relative expression levels of PgCAT in each group after cold stress were 3.712 and 13.181, respectively, indicating an increase compared to the expression levels at room temperature. Plants numbered '10-49', '20-1', '20-18', '20-19', and '20-26' exhibited relatively high PgCAT levels after cold stress. Chlorophyll content is also an important indicator of plant resistance to cold stress. Cold stress damages thylakoid structure and weakens plant photosynthesis. At room temperature, the mean expression level of PgCLH in the ARTP-treated group was higher than in the CK group. The content of chlorophyll synthesis-related genes decreased after cold stress. The mean relative expression levels of PgCLH in the irradiated group were 2.341 and 7.295, respectively, but plants numbered '20-18', '20-19', and '20-23' exhibited relatively high PgCLH expression. After cold stress, the mean expression levels of PgNHX in the 10-min ARTP and 20-min ARTP treatment groups decreased by 67.56% and 51.73%, respectively, compared to those at room temperature. However, the relative expression levels in plants numbered '20-19' and '20-23' were higher. At room temperature, the mean relative expression levels of PgP5CS in the 10-min ARTP and 20-min ARTP treatment groups were 0.809 and 0.968, respectively. After cold stress, the mean relative expression levels of PgP5CS in the 10-min ARTP and 20-min ARTP mutagenesis groups were 8.1 and 6.7 times higher than those in the CK group, respectively.

[0224] (2-2) Principal component and membership function analysis of cold-resistant potential plant phenotypes and physiological indicators

[0225] Principal component analysis (PCA) was used to reduce the dimensionality of the phenotypic and physiological indicators of cold-tolerant plants. Six principal components were extracted, with a cumulative contribution rate of 79.65%, fully reflecting the original data. The following are the main characteristics of each principal component: In the first principal component (F1), morphological indicators such as PgCLH (0.799), leaf width (0.864), PgNHX (0.729), plant height (0.758), tiller number (0.678), and main stem diameter (0.656) had high positive loadings, while relative conductivity (-0.651) and catalase (-0.422) showed high negative loadings, indicating that F1 primarily reflects the negative correlation between plant growth morphology and conductivity and catalase activity. The second principal component (F2) was dominated by proline (0.837) and soluble sugars (0.813), with significant contributions from total chlorophyll (0.561) and peroxidase (0.496), indicating that F2 was primarily associated with osmotic regulators (proline and soluble sugars) and photosynthetic pigment content. The third principal component (F3) showed high negative loadings for SOD (-0.454) and PgCAT (-0.579), while leaf length (0.452) and EC (0.465) showed positive correlations. The fourth principal component (F4) was primarily dominated by malondialdehyde (0.671), PgP5CS (0.529), and peroxidase (0.461), while total chlorophyll (-0.724) showed a high negative loading. The main contributing indicators of the fifth principal component (F5) were superoxide dismutase (0.650) and tiller number (0.438), while PgNHX (-0.45), PgCLH (-0.424), and internode length (-0.332) showed negative loadings. The sixth principal component (F6) was mainly contributed by PgNHX (0.320), soluble sugars (0.272), and superoxide dismutase (0.263), while PgP5CS (-0.462) and POD (-0.327) showed negative loadings.

[0226] Principal component analysis (PCA) was used to comprehensively evaluate the phenotypic and physiological parameters of 17 plants with potential cold tolerance (including the control, CK) after cold stress. Principal component factor scores (F1-F6) and comprehensive scores (D values) were calculated for each sample, and the samples were ranked by D value to assess their cold tolerance. The top five plants with potential cold tolerance: plant '20-23' (D = 0.706) performed exceptionally well in the F1 (morphological characteristics), F2 (osmotic regulation and photosynthetic pigments), and F6 (tillering structure). '20-22' (D = 0.633) had the highest score in the F3 (osmotic regulation) and also had high antioxidant activity in the F5. '20-18' (D = 0.572) had the highest score in the F5 (antioxidant activity) and also had high morphological characteristics in the F1. '10-48' (D = 0.539) performed best in the F2 (photosynthetic pigments) and F4 (membrane stability). '10-49' (D = 0.514) scored high in the F3 (osmotic adjustment) index, with other factors relatively balanced. The CK group ranked 14th (D = 0.333) and showed weak cold tolerance, indicating that the selected plants with potential for cold tolerance significantly outperformed common varieties. '20-1' (D = 0.242) and '20-26' (D = 0.283) performed poorly on all principal components, with the weakest cold tolerance (Table 8).

[0227] The top 10 cold-resistant plants selected by semi-lethal temperature are: '20-19'>'20-18'>'20-25'>'20-23'>'20-1'>'10-36'>'20-22'>'20-27'>'20-28'>'20-26'. The top 10 cold-resistant plants selected by membership function are: '20-23'>'20-22'>'20-18'>'10-48 The results showed that '>'10-49'>'20-24'>'20-19'>'10-36'>'10-17'>'20-28', and 6 plants showed outstanding cold resistance in both screening methods, namely '20-18', '20-19', '20-22', '20-23', '20-28' and '10-36', with only slight differences in ranking, indicating that both methods can achieve effective screening of cold-resistant germplasm (Table 9).

[0228] Table 8. Principal component analysis of ARTP cold-resistant potential plant phenotypes and various cold-resistant physiological indicators

[0229]

[0230] Table 9. Comprehensive evaluation of phenotypes and physiological indicators of cold-resistant plants after cold stress

[0231]

[0232]

[0233] Note: The larger the comprehensive evaluation D value, the better the cold resistance. The D values are sorted from large to small.

[0234] 7. Mutagenic effects of ARTP radiation treatment on field indicators of cold-resistant potential plants of 'Bonde No. 1'

[0235] (1) Mutagenic effects of ARTP radiation treatment on field phenotypic indicators of cold-resistant potential plants of 'Bonde No. 1'

[0236] From Table 10, Figure 18-23 Plant heights in the 10-minute ARTP treatment group ranged from 61.30 to 96.70 cm, with an average of 77.91 cm. Variety '10-49' had the highest plant height. Variety '20-28' had the highest plant height, with a range of 36.80 to 93.20 cm, with an average of 94.63 cm. The 20-minute ARTP treatment group also had the highest coefficient of variation for plant height, with both positive and negative variation rates of 20%. The average number of tillers in the 10-minute ARTP treatment group was 8.29, with both positive and negative variation rates of 14.29%. The average number of tillers in the 20-minute ARTP treatment group was 13.70, with positive and negative variation rates of 30% and 20%, respectively. The average internode length in the 10-minute ARTP and 20-minute ARTP treatment groups increased by 11.71% and 41.47%, respectively, compared to the CK group. The mean main stem diameters for the CK, 10-min ARTP, and 20-min ARTP treatments were 1.88 cm, 1.43 cm, and 1.76 cm, respectively, with positive variation rates of 23.81% and 23.33%, respectively. The main stem diameters of cultivars 20-22 and 20-23 increased significantly compared to the CK group. The mean leaf lengths for the 10-min ARTP and 20-min ARTP treatments decreased by 14.41% and 5.02%, respectively, compared to the CK group. However, the leaf lengths of cultivars 20-23, 10-49, and 20-18 were significantly longer than those of the CK group. The leaf width for the 20-min ARTP treatment group had a positive variation rate of 16.67%, with cultivars 20-18, 20-22, and 20-23 showing significantly greater leaf widths than the CK group.

[0237] Table 10. Field phenotypic observation of cold-resistant potential plants

[0238]

[0239]

[0240] (2) Mutagenic effects of ARTP radiation treatment on field quality indicators of cold-resistant potential plants of 'Bonde No. 1'

[0241] (2-1) Mutagenic Effects of ARTP Radiation Treatment on ADF Content in Cold-Tolerant Potential Plants of 'Bangde No. 1'

[0242] Depend on Figure 24 A shows that the mean ADF content in the 10-min ARTP and 20-min ARTP treatment groups increased by 7.02% and 11.08% respectively compared with that in CK. Figure 24 B shows that only the ADF content of '10-45' is lower than the CK value. The ADF content of plants numbered '10-48', '20-18', '20-28', '20-23' and '10-36' has no significant difference compared with the CK. The ADF content of plants numbered '20-26', '20-24' and '20-19' is significantly higher than that of the CK group (P < 0.05), which are 1.1576 times, 1.1784 times and 1.1982 times of that of the CK group, respectively.

[0243] (2-2) Mutagenic Effects of ARTP Radiation Treatment on NDF Content in Cold-Tolerant Potential Plants of 'Bangde No. 1'

[0244] Depend on Figure 25 A shows that the mean NDF content of the CK, 10min ARTP, and 20min ARTP treatment groups were 0.5192, 0.5688, and 0.5720, respectively. The mean NDF content of the 10min ARTP and 20min ARTP treatment groups increased by 9.55% and 10.17% compared with that of the CK. Figure 25 B shows that only the mean NDF content of '10-45' is lower than that of CK. The NDF contents of plants '10-48', '20-23', '20-28', '20-22', '20-27', '10-28' and '10-36' are slightly higher than those of the CK group. The NDF contents of '10-49' and '10-17' are significantly higher than those of the CK group, and their NDF values increased by 19.34% and 19.43% respectively compared with the CK group.

[0245] (3) Mutagenic effect of ARTP radiation treatment on protein content in cold-resistant plants of 'Bangde No. 1'

[0246] Depend on Figure 26 A shows that the mean protein content of the CK, 10min ARTP, and 20min ARTP treatment groups were 21.084%, 19.399%, and 21.136%, respectively. The mean protein content of the 10min ARTP treatment group was 7.99% lower than that of the CK, but there was no significant difference among the treatment groups. Figure 26 B shows that the protein content of plants '20-23', '20-28', '20-26', '20-18', '20-22' and '20-25' was slightly higher than that of the CK group.

[0247] (4) Mutagenic effect of ARTP radiation treatment on chlorophyll content in cold-resistant plants of 'Bonde No. 1' before and after frost

[0248] After the Frost Descent, the temperature drops sharply to below 0℃. The plant suffers from severe water shortage and the leaf color darkens. The leaves become dry, thin and wrinkled, and a large number of leaves are bent. Only some young leaves inside the base are not frozen to death. The stems lose water and become brittle and easy to fall over. There is liquid oozing out of the stem nodes, and the plant is brown as a whole.

[0249] Depend on Figure 27 A shows that before frost, the mean total chlorophyll content of the CK, 10min ARTP and 20min ARTP treatment groups were 1.28mg / g, 1.41mg / g and 1.24mg / g, respectively. The mean total chlorophyll content of the 10min ARTP treatment group before frost was 1.105 times that of the CK group. The total chlorophyll content of the 20min ARTP treatment group before frost was 3.34% lower than that of the CK group. However, there was no significant difference in the mean total chlorophyll content among the treatment groups. After frost, the total chlorophyll content of the CK, 10min ARTP and 20min ARTP treatment groups were 0.46mg / g, 1.10mg / g and 0.97mg / g, respectively, which were 64.22%, 22.17% and 21.89% lower than those before frost, respectively. Figure 27 B shows that, except for the CK group and '20-1', the total chlorophyll content decreased significantly before and after frost (P < 0.05), the total chlorophyll content of the other plants after frost was slightly lower than that before frost.

[0250] (5) Comprehensive analysis of the field mutagenic effects of ARTP radiation treatment on cold-resistant potential plants of 'Bonde No. 1'

[0251] (5-1) Principal component analysis of field phenotypes and physiological indicators of plants with cold-resistant potential

[0252] Principal component analysis identified three principal components, with a cumulative contribution rate of 77.345%. Plant height (0.807), tillering (0.734), leaf length (0.794), main stem diameter (0.774), and leaf width (0.922) had high positive loadings on the first principal component, indicating that the first principal component primarily reflects the plant's growth morphology and structural characteristics. Chlorophyll (0.658) and protein (0.501) had high loadings on the second principal component, but NDF (-0.816) and ADF (-0.770) showed significant negative correlations on the second principal component. Protein (0.762) had the highest loading on the third principal component (Table 11).

[0253] (5-2) Analysis of membership functions of field phenotypes and physiological indicators of cold-resistant potential plants

[0254] The membership function analysis of field phenotypic and physiological indicators of cold-resistant plants was carried out. The top 10 plants were ranked as '20-22' > '20-23' > '20-18' > '20-25' > '10-49' > '20-24' > '20-26' > '10-17' > '20-28' > '10-48'. Combined with the cold-resistant plants selected by semi-lethal temperature and membership function analysis method: '20-18', '20-19', '20-22', '20-23', '20-28' and '10-36', '20-18', '20-22', '20-23' and '20-28' were further selected as high-quality cold-resistant plants (Table 12).

[0255] Table 11. Principal component analysis of field phenotypes and physiological indicators of plants with cold-resistant potential

[0256]

[0257] Table 12. Membership function analysis of field phenotypes and physiological indicators of plants with cold-resistant potential

[0258]

[0259] The present invention has been described in detail above. It will be apparent to those skilled in the art that the present invention may be practiced over a wide range of parameters, concentrations, and conditions without departing from the spirit and scope of the present invention and without unnecessary experimentation. Although specific embodiments have been given herein, it should be understood that further modifications may be made to the present invention. In summary, this application is intended to encompass any variations, uses, or improvements to the present invention, including those made by conventional techniques known in the art that depart from the scope of the present invention. Applications of the essential features may be made within the scope of the following claims.

Claims

1. A plasma mutagenesis method for 'Bangde No. 1' hybrid Pennisetum callus, comprising the following steps: subjecting 'Bangde No. 1' hybrid Pennisetum callus to ARTP radiation mutagenesis; the ARTP radiation mutagenesis time is 10-20 minutes.

2. The method according to claim 1, wherein: The ARTP radiation mutagenesis time is 20 min.

3. The method according to claim 1 or 2, characterized in that: The method also includes the step of inducing culture of 'Bangde No. 1' hybrid Pennisetum seeds to obtain 'Bangde No. 1' hybrid Pennisetum callus before ARTP radiation mutagenesis.

4. A plasma mutagenesis breeding method for 'Bangde No. 1' hybrid Pennisetum, comprising the following steps: subjecting 'Bangde No. 1' hybrid Pennisetum callus to ARTP radiation mutagenesis according to the method of any one of claims 1 to 3 to achieve mutagenesis breeding.

5. The method according to claim 4, characterized in that: The method further comprises the step of screening cold-resistant plants after the ARTP radiation mutagenesis.

6. The method according to claim 4 or 5, characterized in that: The method comprises the following steps: 1) ARTP radiation mutagenesis was performed on the callus of 'Bangde No. 1' hybrid Pennisetum to obtain the mutagenized callus; 2) performing differentiation culture on the induced callus to obtain differentiated budding tissue; 3) performing rooting culture on the differentiated sprouted tissue to obtain rooted seedlings; 4) transplanting the rooted seedlings into soil for cultivation to obtain regenerated seedlings; 5) subjecting the regenerated seedlings to low temperature treatment to obtain cold-resistant plants through screening.

7. The method according to claim 6, characterized in that: In the step 5), the low-temperature treatment is carried out at 0° C. for 1 day.

8. The method according to claim 6 or 7, characterized in that: In the step 5), the cold-resistant plants are screened according to the semi-lethal temperature or the membership function analysis method.

9. Any application of a1) to a10) below: a1) Use of the method according to any one of claims 1 to 8 in promoting callus differentiation of Pennisetum hybridum; a2) Use of the method according to any one of claims 1 to 8 in promoting rooting of hybrid Pennisetum callus; a3) Use of the method according to any one of claims 1 to 8 in promoting the formation of hybrid Pennisetum callus seedlings; a4) Use of the method according to any one of claims 1 to 8 in increasing the number of hybrid Pennisetum plants; a5) Use of the method according to any one of claims 1 to 8 for increasing the tillering number of hybrid Pennisetum; a6) Use of the method according to any one of claims 1 to 8 for increasing the leaf size of hybrid Pennisetum; a7) Use of the method according to any one of claims 1 to 8 in regulating the content of osmotic regulating substances in hybrid Pennisetum; a8) Use of the method according to any one of claims 1 to 8 in regulating the antioxidant enzyme activity of hybrid Pennisetum; a9) Use of the method according to any one of claims 1 to 8 in improving the cold resistance of hybrid Pennisetum; a10) Use of the method according to any one of claims 1 to 8 in preparing new cold-resistant hybrid Pennisetum germplasm.

10. The use according to claim 9, characterized in that: Increasing the size of hybrid Pennisetum leaves is embodied by increasing the length and / or width of hybrid Pennisetum leaves; Alternatively, the regulation of the content of osmotic regulating substances in the hybrid Pennisetum is manifested in reducing the malondialdehyde content in the hybrid Pennisetum and / or increasing the soluble sugar content in the hybrid Pennisetum; Alternatively, the regulation of the antioxidant enzyme activity of hybrid Pennisetum is manifested in increasing the CAT enzyme activity of hybrid Pennisetum.

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