Method for rapidly screening American pennisetum germplasm with freezing-resistant potential

Abstract

The invention discloses a method for rapidly screening a Pennisetum americanum germplasm with freezing-resistant potential, and relates to the related fields of plant physiology and molecular biology. The method comprises the following steps: (1) preparing germplasm resource plantlets of Pennisetum americanum; (2) cold domestication at 2 DEG C; (3) freezing treatment at-7 DEG C; and (4) recovering at high temperature of 35-40 DEG C. The method has the following advantages and positive effects: firstly, the low-temperature resistance of the pennisetum americana is enhanced through cold domestication, and the difference of different resources to low-temperature response is amplified; freezing the Pennisetum americanum germplasm at-7 DEG C for 5 hours, and starting difference of response of different resources to low temperature; the high-temperature-resistant mechanism of the pennisetum alopecuroides is utilized to assist the pennisetum alopecuroides germplasm to quickly recover the regeneration capacity, and the difference of different resources on low-temperature response is further amplified, so that the pennisetum alopecuroides germplasm with freezing-resistant potential is quickly and efficiently screened.

Description

Technical Field

[0001] The invention relates to the fields related to plant physiology and molecular biology, and in particular to a method for rapidly screening Pennisetum americanum germplasm with freezing resistance potential. Background Art

[0002] Forage is the staple food for livestock and the basis for large-scale intensive farming. American Pennisetum glaucum (L.) R.Br., syn. Cenchrus americanus (L.) Morrone is an annual high-biomass C4 forage grass of the genus Pennisetum in the Poaceae family. It can be used for the breeding of herbivorous poultry and herbivorous freshwater fish. American Pennisetum originated in Africa and has the heat-resistant trait of growing well in high temperature environments. However, it grows poorly when the temperature is below 15°C, and withers and dies as the temperature continues to drop. Because it cannot survive the winter, it is limited to its cultivation in areas such as the North China Plain in my country. Therefore, screening American Pennisetum germplasm with freezing resistance potential will help cultivate low-temperature resistant varieties of American Pennisetum.

[0003] So far, there is no report in the prior art on a method for screening Pennisetum americanum germplasm with freezing resistance potential. Summary of the invention

[0004] The purpose of the present invention is to overcome the problems and shortcomings of the prior art, and to provide a method for rapidly screening Pennisetum germplasm with freezing resistance potential, so as to facilitate the research on low temperature resistance breeding of Pennisetum.

[0005] The object of the present invention is achieved in that:

[0006] The method comprises the following steps:

[0007] ① Preparation of Pennisetum americanum germplasm resources seedlings

[0008] One-month-old Pennisetum fasciatus germplasm seedlings, hybrid breeding seedlings, or gene-editing breeding seedlings are cultivated outdoors at about 35°C;

[0009] ②2℃ cold acclimation

[0010] The material was moved to a low-temperature light incubator and cold-acclimated for 22 hours at 2°C with 12 hours of light;

[0011] ③ Freezing at -7℃

[0012] Set the temperature in the low-temperature light incubator to -7°C and freeze under light for 5 hours;

[0013] ④35-40℃ high temperature recovery

[0014] Move the plant materials that have been frozen for 6 hours directly outdoors to resume growth, at 35-40℃ at noon, and screen the germplasm resources with sprouting new buds.

[0015] Working principle:

[0016] Pennisetum is resistant to high temperatures but not low temperatures. Studies have found that high temperatures can significantly increase the survival rate of Pennisetum seedlings under low temperature stress. In order to screen out germplasm resources with potential for freezing resistance and provide useful resources for low temperature breeding of Pennisetum resources, this method designs a combination treatment method of three groups of temperatures, which amplifies the differences in freezing resistance of different resources before and after freezing treatment, thereby realizing rapid screening of Pennisetum germplasm with potential for freezing resistance. Before freezing treatment, the cold acclimation mechanism is used to amplify the differences in the responses of different resources to low temperatures. The differences in the responses of different resources to low temperatures are initiated through freezing treatment. After freezing treatment, the high temperature resistance mechanism of Pennisetum is used to further amplify the differences in the responses of different resources to low temperatures, thereby quickly and efficiently screening Pennisetum germplasm with potential for freezing resistance.

[0017] The purpose of this method:

[0018] By amplifying the differences in the freezing resistance of germplasm resources, it can be used to quickly and efficiently screen germplasm with freezing resistance potential from American Pennisetum germplasm; it can also be used by breeders to quickly and efficiently screen out individual plants with freezing resistance in the breeding of American Pennisetum for freezing resistance traits.

[0019] This method has the following advantages and positive effects:

[0020] First, the low-temperature tolerance of Pennisetum was enhanced through cold acclimation, amplifying the differences in the responses of different resources to low temperatures; the Pennisetum germplasm was frozen at -7℃ for 5 hours to initiate the differences in the responses of different resources to low temperatures; the high-temperature resistance mechanism of Pennisetum was used to assist the Pennisetum germplasm to quickly restore its regeneration ability, further amplifying the differences in the responses of different resources to low temperatures, thereby quickly and efficiently screening Pennisetum germplasm with freeze-resistant potential. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 : After low temperature stress treatment, it was found that high temperature significantly increased the survival rate of Pennisetum americanum seedlings

[0022] AB: growth status of seedlings (5-6 leaves) before treatment; CE: after treatment at 1°C for 24 hours, recovery at 15°C for 3 hours, recovery at 15°C, 25°C and 35°C for 45 hours respectively, and then recovery in a 25°C culture room for 2 weeks, the growth status and survival rate (E), with 24 seedlings in each replicate and 4 replicates for each temperature treatment.

[0023] Figure 2 This is a flow chart for screening Pennisetum truncatum resources with frost resistance potential. In the figure:

[0024] 1—Strong seedlings with 7-8 leaves grown outdoors in summer;

[0025] Cold acclimation treatment at 2-2℃ for 22 hours;

[0026] Freeze at 3-7℃ for 5 hours;

[0027] 4—35-40℃ summer high temperature outdoor recovery;

[0028] 5—Identify plantings with freeze tolerance potential.

[0029] Figure 3 : The Pennisetum americanum germplasm with frost resistance potential screened by this method, in the figure:

[0030] A: Utilization Figure 2 Process -7℃ treatment for 5 hours and then moved to the outdoor Pennisetum resources to restore the growth status after 0 and 3 days;

[0031] B: Utilization Figure 2 The three germplasms with frost resistance potential selected by the process recovered their growth status before treatment, 7 days, 14 days and 40 days after treatment. DETAILED DESCRIPTION

[0032] The following is a detailed description with reference to the accompanying drawings and embodiments:

[0033] ① Preparation of Pennisetum americanum germplasm resources seedlings

[0034] In summer, the American Pennisetum germplasm resource seedlings were cultivated outdoors (around 35-40℃ at noon). The seedlings were about one month old, had not yet jointed, and were sturdy seedlings with 7-8 leaves.

[0035] ②2℃ cold acclimation

[0036] After the temperature of the low-temperature light incubator reaches 2°C, quickly move the materials into the incubator and cold acclimate for 22 hours (12 hours of light).

[0037] ③ Freezing at -7℃

[0038] After the temperature in the low-temperature light incubator was set to -7°C, the timer was started and the freezing treatment was performed for 5 hours.

[0039] ④35-40℃ high temperature recovery

[0040] Move the plant materials that have been frozen for 6 hours outdoors to resume growth (35-40℃ at noon), pay attention to replenishing water in time, and select germplasm resources that can sprout new buds.

Claims

1. A method for rapidly screening Pennisetum fasciatum germplasm with freezing resistance potential, characterized in that The following steps are involved: ① Preparation of Pennisetum americanum germplasm resources seedlings Outdoors, at about 35℃ at noon, the American Pennisetum germplasm resource seedlings were cultivated, and the seedlings were 1 month old; ②2℃ cold acclimation The material was moved to a low-temperature light incubator and cold-acclimated for 22 hours at 2°C with 12 hours of light; ③ Freezing at -7℃ Set the temperature in the low-temperature light incubator to -7°C and freeze under light for 5 hours; ④35-40℃ high temperature recovery Move the plant materials that have been frozen for 6 hours directly outdoors, at 35-40℃ at noon, to resume growth and screen the germplasm resources with sprouting new buds.

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