Phyllostachys edulis mutagenesis breeding method based on artp

Abstract

The present application relates to the technical field of plant breeding, and in particular to a mutation breeding method for Phyllostachys edulis based on ARTP. The mutation breeding method comprises: using atmospheric room temperature plasma to treat the seeds of Phyllostachys edulis, and the parameters include: radio frequency power of 200-400 W, radiation distance of 2-3 mm, and treatment time of 8-55 minutes. The present application studies a mutation breeding method suitable for Phyllostachys edulis, and through a specific degree of ARTP mutation of the seeds of Phyllostachys edulis, the number of tillers of the seedlings of Phyllostachys edulis can be increased and the yellowing rate of the seedling of Phyllostachys edulis can be reduced under the condition of ensuring a certain seedling rate. The mutation breeding method provided by the present application can be used for breeding Phyllostachys edulis varieties with excellent traits, and has important value in the field of Phyllostachys edulis breeding.

Description

Technical Field

[0001] This invention relates to the field of plant breeding technology, and in particular to a method for mutagenesis breeding of moso bamboo based on ARTP. Background Technology

[0002] Mutation is the source of most genetic variations and the driving force behind species evolution in nature. Plant mutation breeding refers to a breeding method that uses physical radiation or chemical treatment to induce variations in the genetic characteristics of plants, and then selects individual plants / individuals that meet specific requirements from the mutated population to cultivate new varieties or germplasm. Mutation refers to a method of generating gene mutations by using external factors to cause base mismatches and recombination in DNA molecules within cells.

[0003] Most mutations are harmful; random mutations can disrupt normal gene function, leading to harmful or detrimental traits. However, after mutagenesis, technicians may still obtain a very small number of beneficial mutations. These mutations are invaluable genetic resources for increasing population genetic diversity and enhancing breeding potential. There are many types of mutagenesis, including physical mutagenesis such as radiation mutagenesis and ultraviolet mutagenesis, chemical mutagenesis such as EMS or MMS, and biological mutagenesis such as transposon mutagenesis and CRISPR / Cas9 gene editing.

[0004] Moso bamboo (Phyllostachys edulis) is a scattered bamboo species belonging to the genus Phyllostachys in the family Poaceae (Barnhart). It has a long history of cultivation, a large distribution area, high stock volume, high output value, in-depth basic and applied research, and the best degree of industrial utilization. As a superior bamboo species with good ecological and social benefits, integrating timber, food, ornamental, and ecological uses, moso bamboo plays an important role in solving problems such as ecological degradation and resource scarcity. With the proposal of the "bamboo-for-plastic" initiative, the application prospects of bamboo have been greatly enriched, while also placing higher demands on the diversity of bamboo resources. There is a need for bamboo species with higher biomass, varying strength and toughness, and other diverse traits to meet different application scenarios in fields such as disposable products, daily necessities, construction and transportation, and engineering facilities.

[0005] Due to excessive pressure from artificial cultivation and breeding, the genetic diversity of moso bamboo germplasm resources has gradually decreased, leading to smaller differences between plants and a decline in traits such as adaptability and resistance. Moreover, because bamboo has a long vegetative growth cycle, low fruit set rate, and the entire plant dies after flowering and fruiting, traditional hybridization breeding is difficult to achieve, transgenic systems are extremely difficult to establish, and breeding methods are extremely limited. This makes it difficult to select and cultivate superior varieties of moso bamboo for timber, shoots, and pulp production. Summary of the Invention

[0006] To address the problems existing in the prior art, this invention provides a method for mutagenesis breeding of moso bamboo based on ARTP. This method overcomes the shortcomings of existing bamboo selection and breeding techniques, such as the single source of the breeding population, low genetic diversity, limited available genetic variation resources, and low breeding efficiency, by using atmospheric pressure and room temperature plasma radiation mutagenesis under specific conditions.

[0007] In a first aspect, the present invention provides a method for mutagenesis of moso bamboo, comprising: treating moso bamboo seeds with atmospheric pressure room temperature plasma, wherein the parameters include: radio frequency power of 200-400W, radiation distance of 2-3mm, and treatment time of 8-55 minutes.

[0008] Furthermore, the bamboo seeds are either bamboo seeds with exposed white hairs or bamboo seeds without exposed white hairs.

[0009] Furthermore, for bamboo seeds with white tips, the treatment time is 8–55 minutes; and / or for bamboo seeds without white tips, the treatment time is 8–40 minutes.

[0010] Furthermore, for bamboo seeds with white tips, the treatment time is 10–55 minutes; and / or for bamboo seeds without white tips, the treatment time is 10–40 minutes.

[0011] Furthermore, during the atmospheric pressure and room temperature plasma treatment, the working environment is helium, and the helium flow rate is 10-20L; after 10 minutes of treatment, water is replenished every 8-12 minutes.

[0012] Furthermore, after the atmospheric pressure and room temperature plasma treatment, the mutagenized bamboo seeds are transplanted and cultivated.

[0013] Furthermore, the colonization culture includes: The mutagenized bamboo seeds were transplanted and cultured under the following conditions: temperature 24-28℃, and the ratio of dark culture time to light culture time was 1:(1.8-2.2).

[0014] Furthermore, the planting in the soil includes: planting in the water of a germination bag, or planting in the soil of a seedling tray.

[0015] Furthermore, the soil comprises nutrient soil and vermiculite in a mass ratio of (1-2):(1-2).

[0016] Secondly, the present invention provides a method for breeding moso bamboo, comprising: performing mutation breeding using the method described above.

[0017] Thirdly, the present invention provides the application of the mutagenesis method described above in increasing the number of tillers or reducing the yellowing rate of moso bamboo.

[0018] The present invention further provides the application of the mutagenesis method in cultivating moso bamboo varieties with high tiller number or low yellowing rate.

[0019] The present invention has the following beneficial effects: This invention presents an ambient pressure, room temperature plasma mutagenesis method suitable for moso bamboo breeding. Through ARTP treatment under specific conditions, the number of tillers in moso bamboo can be increased while maintaining a certain seedling emergence rate, and the yellowing rate can be reduced. This mutagenesis method can be applied to the trait improvement breeding of moso bamboo to cultivate bamboo with superior traits, and has significant application value. Attached Figure Description

[0020] To more clearly illustrate the technical solutions in this invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of this invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0021] Figure 1 These are diagrams of bamboo seeds with and without white spots, as well as an improved sample carrier, provided in Embodiment 1 of the present invention; the upper left diagram shows bamboo seeds with white spots, and the lower left diagram shows bamboo seeds without white spots.

[0022] Figure 2 This is a statistical result chart of the seedling rate and tiller number of moso bamboo under different mutagenesis times provided in Embodiment 1 of the present invention.

[0023] Figure 3 This is a statistical result chart of the bamboo seedling rate and yellowing rate under different mutagenesis times provided in Embodiment 2 of the present invention. Detailed Implementation

[0024] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions of this invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some, not all, of the embodiments of this invention. All other embodiments obtained by those skilled in the art based on the embodiments of this invention without creative effort are within the scope of protection of this invention.

[0025] Unless otherwise specified, the experimental methods involved in the following embodiments are conventional methods in the art. For example, you can refer to the experimental manual in the art or follow the conditions recommended in the manufacturer's instructions.

[0026] Unless otherwise specified, all experimental materials and reagents used in the following examples are commercially available.

[0027] Example 1

[0028] This invention provides a method for mutagenesis of moso bamboo seeds using ambient pressure room temperature plasma method (hereinafter referred to as ARTP), comprising the following steps: 1. Seed sample germination

[0029] Select 2000 bamboo seeds with the outer shell removed, soak them in clean water at room temperature for 2-3 days, changing the water every 12 hours, and removing the poor-quality seeds that float on the surface.

[0030] 2. Preparation of mutagenesis seed samples

[0031] Place the germinated bamboo seeds on a germination tray lined with absorbent paper to encourage the emergence of white seeds. Cover the seeds with absorbent paper and spray with ddH2O to maintain moisture. Spray with ddH2O every 2 days and remove any contaminated seeds.

[0032] 3. The mutagenic seeds were loaded into the sample carrier.

[0033] To increase the single-shot mutation flux, the sample carrier was redesigned (e.g., Figure 1 As shown, the seeds were placed vertically for mutagenesis treatment. While ensuring that the seeds remained moist during the mutagenesis process, the mutagenesis throughput was increased from 40-50 seeds / time to 180-200 seeds / time. The seeds were inserted into the sample carrier with tweezers, and in one operation, half of the seeds and half of the seeds that had not yet shown white were selected for mutagenesis treatment.

[0034] 4. Set the parameters of the ARTP mutation breeding instrument: the working gas is high-purity helium, the radio frequency power is 400W, the helium flow rate is 15L / min, and the distance is 3mm.

[0035] 5. Mutagenesis process

[0036] After the sample carrier was fully hydrated, it was placed on the stage of the ARTP mutation breeding instrument to begin mutagenesis. The mutagenesis times for different groups were set to 5, 10, 20, 30, and 50 min, respectively. For the mutagenesis groups with radiation time exceeding 10 min, the instrument was paused at 10, 20, 30, and 40 min, and 1 mL of ddH2O was added. The preparation for the control group (0 min) was the same as that for the mutagenesis group. After transferring the sample carrier, it was left to stand for 2 min without mutagenesis treatment.

[0037] Seeds from the control group and the mutation group were inserted into germination bags, 50 mL of ddH2O was added to the germination bags, and the bags were covered with black plastic bags to simulate a dark environment. The bags were then placed in an artificial climate chamber and cultured at a constant temperature of 24°C under a light-to-dark cycle of 16 h and 8 h. The water was changed every 7 days and contaminated seeds were removed.

[0038] Seed germination and seedling growth were continuously observed during the cultivation period, and the seedling survival rate was calculated at 40 days.

[0039] 6. Results of mutagenesis

[0040] The results are as follows Figure 2 As shown, the survival rate of moso bamboo seeds decreased with the extension of treatment time. The seedling rate of moso bamboo seeds that had not yet sprouted white hairs was higher than that of moso bamboo seeds that had sprouted white hairs after 50 minutes of treatment. At 50 minutes, the seedling rate of moso bamboo seeds that had not yet sprouted white hairs was 53.33%, while that of seeds that had sprouted white hairs was 21.11%.

[0041] Furthermore, when the treatment time exceeded 10 minutes, the number of tillers in moso bamboo seeds increased significantly, with an even more significant increase in the number of white-tillered seeds.

[0042] Example 2

[0043] This invention provides a method for inducing mutagenesis in bamboo seeds using ambient pressure and room temperature plasma, comprising the following steps: 1. Germination of seed samples.

[0044] Select 2000 bamboo seeds with the outer shell removed, soak them in clean water at room temperature for 2-3 days, changing the water every 12 hours, and removing the poor-quality seeds that float on the surface.

[0045] 2. Preparation of mutagenesis seed samples

[0046] Place the germinated bamboo seeds on a germination tray lined with absorbent paper to encourage the emergence of white seeds. Cover the seeds with absorbent paper and spray with ddH2O to maintain moisture. Spray with ddH2O every 2 days and remove any contaminated seeds.

[0047] 3. The mutagenic seeds were loaded into the sample carrier.

[0048] Use tweezers to insert the bamboo seeds into the sample carrier holes, being careful not to scratch the seed coat, otherwise the bamboo seeds are very prone to fungal growth. In one operation, select half of the seeds with white sprouts and half of the seeds without white sprouts for mutagenesis treatment.

[0049] 4. Set the parameters of the ARTP mutation breeding instrument: the working gas is high-purity helium, the radio frequency power is 400W, the helium flow rate is 15L / min, and the distance is 3mm.

[0050] 5. Mutagenesis process: After the sample carrier is fully hydrated, it is placed on the stage of the ARTP mutagenesis breeding instrument to start mutagenesis. The mutagenesis time for different groups is set to 5, 10, 20, 30 and 50 min respectively. For the mutagenesis groups with radiation time exceeding 10 min, the instrument is paused at 10, 20, 30 and 40 min respectively, and 1 mL ddH2O is added. The preparation of the control group (0 min) is the same as that of the mutagenesis group. After transferring to the sample carrier, it is left to stand for 2 min without mutagenesis treatment.

[0051] Seeds from the control group and the mutation group were planted in seed trays. The substrate consisted of nutrient soil and vermiculite in a 1:1 ratio (by mass). The seed trays were placed in an artificial climate chamber and cultured under 16 hours of light and 8 hours of darkness, with regular watering.

[0052] Seed germination and seedling growth were continuously observed during the cultivation period, and the seedling survival rate was calculated at 40 days.

[0053] 6. Results of mutagenesis

[0054] The results are as follows Figure 3 As shown, the seedling rate of bamboo seeds with white tips did not change significantly with the extension of treatment time, while the seedling rate of bamboo seeds without white tips was close to 50% after treatment for 30 min and 50 min. In addition, the yellowing rate of seedlings with non-white tips decreased significantly after treatment for 10 min and 20 min, while the yellowing rate of seedlings with white tips decreased after treatment for 10 min and decreased significantly after treatment for 30 min and 50 min.

[0055] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; 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; and these 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 inducing mutation in moso bamboo, characterized in that, include: Moso bamboo seeds were treated with atmospheric pressure room temperature plasma with parameters including: radio frequency power of 200~400W and radiation distance of 2~3mm. The bamboo seeds are either bamboo seeds with exposed white hairs or bamboo seeds without exposed white hairs. For bamboo seeds with white tips, the treatment time is 10-55 minutes; for bamboo seeds without white tips, the treatment time is 10-30 minutes.

2. The mutagenesis method according to claim 1, characterized in that, During the atmospheric pressure and room temperature plasma treatment process, the working environment is helium, and the helium flow rate is 10~20L; and / or, After a 10-minute treatment, replenish the water every 8-12 minutes.

3. The mutagenesis method according to claim 1 or 2, characterized in that, After atmospheric pressure and room temperature plasma treatment, the mutagenized bamboo seeds were transplanted and cultured.

4. The mutagenesis method according to claim 3, characterized in that, The colonization culture includes: The mutagenized bamboo seeds were planted in soil for cultivation at a temperature of 24-28℃, with a ratio of dark cultivation time to light cultivation time of 1:(1.8-2.2).

5. A method for breeding moso bamboo, characterized in that, include: Mutation breeding is carried out using the method described in any one of claims 1-4.

6. The application of the mutagenesis method according to any one of claims 1-4 in increasing the number of tillers or reducing the yellowing rate of moso bamboo.

7. The application of the mutagenesis method according to any one of claims 1-4 in the cultivation of moso bamboo varieties with high tiller number or low yellowing rate.

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