A method for improving the induction rate of callus of anther culture of Leymus chinensis

Abstract

The application discloses a method for improving the induction rate of callus of anther culture of Leymus mollis, and relates to a method for culturing callus of Leymus mollis. The method comprises the following steps: selecting a young ear of a Leymus mollis hybrid F1 generation plant; performing low-temperature treatment; performing disinfection and cleaning; placing the anther on an induction culture medium; and then performing constant-temperature induction culture in a dark condition to obtain callus. The Leymus mollis haploid is obtained through anther culture, and after doubling, pure breeding material can be quickly obtained, the induction rate of callus can be further greatly improved, the breeding period of Leymus mollis can be shortened, and the breeding efficiency is improved; and a new way is provided for obtaining more high-quality Leymus mollis germplasm resources.

Description

Technical Field

[0001] This invention relates to a method for culturing callus tissue from Leymus chinensis. Background Technology

[0002] Leymus chinensis (Trin.) Tzvel., a perennial grass belonging to the genus Leymus, possesses advantages such as strong salt and alkali tolerance, good palatability, and high crude protein content. However, existing Leymus chinensis varieties are lacking, with few bred varieties and low yields. Currently, the widespread use of seed propagation and division propagation for breeding new Leymus chinensis varieties reflects outdated breeding techniques, resulting in low yields of high-quality germplasm resources and a breeding time of 8-10 years, severely hindering the progress of new Leymus chinensis variety development. Even with tissue culture techniques using seeds and young spikelets as explants, the low callus induction rate and long breeding time for Leymus chinensis remain problems. Summary of the Invention

[0003] The purpose of this invention is to accelerate the breeding of new varieties of Leymus chinensis and to provide a method for improving the callus induction rate of Leymus chinensis anther culture.

[0004] The present invention provides a method for improving the callus induction rate in Leymus chinensis anther culture, comprising the following steps:

[0005] I. Select young spikelets from F1 generation hybrids of different ecotypes of Leymus chinensis;

[0006] 2. Wrap the cut end of the young spike with a damp paper towel to keep it moist, then wrap the entire spike with plastic wrap to prevent moisture loss; then place it in a dark environment at 4℃ for 2-5 days.

[0007] 3. After low-temperature treatment, cut off the flag leaf and stem of the young spikelet, disinfect it with alcohol, then rinse it with sterile distilled water to remove the surface alcohol; then soak it in sodium hypochlorite solution, and then rinse the young spikelet with sterile distilled water.

[0008] 4. In a sterile environment, use tweezers to peel off the flag leaf sheath of the young spikelet, take out the young spikelet, and then use scissors to cut off the spikelets at the top and bottom of the young spikelet, leaving the middle spikelet; take the two outermost florets of the retained spikelet, open the individual florets to expose the anthers inside, carefully pick out the anthers with tweezers and place them on the induction medium, then seal the culture dish and place it in a constant temperature and dark environment at 27±1℃ to induce callus culture.

[0009] In step one, young spikes from the early to mid-heading stage of hybrid F1 generation sheepgrass plants are selected.

[0010] In step one, the parents are either female gray-green type × male yellow type, female yellow-green type × male gray-green type, or female yellow type × male yellow-green type;

[0011] The induction medium in step four is MS medium containing 2.0 mg / L 2,4-dichlorophenoxyacetic acid (2,4-D) + 30 g / L sucrose + 7 g / L agar.

[0012] Furthermore, the young spikelets obtained in step one are inserted with the cut end facing down into a container filled with clean water for preservation and transportation.

[0013] Furthermore, in step one, under sunny and stable weather conditions, the young spikelets are cut at a time between 9:00-10:00 AM and / or 2:00-3:00 PM.

[0014] Furthermore, in step one, the cut of the young spikelet is located at the second-to-last node of the sheepgrass plant.

[0015] Furthermore, the number of intermediate spikelets retained is 6 to 8.

[0016] Furthermore, in step two, the young spikelets are wrapped with 2 to 3 layers of plastic wrap.

[0017] In this invention, the emergence of young spikelets accounting for 1 / 3 of the total spikelet length is the early stage of spikelet emergence in Leymus chinensis plants, and the emergence of young spikelets accounting for 1 / 2 of the total spikelet length is the middle stage of spikelet emergence in Leymus chinensis plants.

[0018] The haploid breeding technology employed in this invention can obtain a rich variety of genetically homozygous breeding materials within two generations, shortening the breeding time for high-quality varieties. It is a rapid approach to optimizing crop breeding and has significant application value. Anther culture is one of the main methods for producing haploids, but it is more challenging than tissue culture using seeds and young spikelets as explants. This invention obtains Leymus chinensis haploids through anther culture, and doubling them can rapidly yield homozygous breeding materials. Furthermore, it can significantly improve the callus induction rate, shortening the breeding cycle of Leymus chinensis and increasing breeding efficiency; providing a new approach to obtaining more high-quality Leymus chinensis germplasm resources. Detailed Implementation

[0019] The technical solutions in the embodiments of the present invention have been clearly and completely described. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0020] It should be noted that, unless otherwise specified, the embodiments and features described in the present invention can be combined with each other.

[0021] Specific Implementation Method 1: This implementation method for improving the callus induction rate in Leymus chinensis anther culture is carried out according to the following steps:

[0022] I. Select young spikelets from F1 generation hybrids of different ecotypes of Leymus chinensis;

[0023] 2. Wrap the cut end of the young spike with a damp paper towel to keep it moist, then wrap the entire spike with plastic wrap to prevent moisture loss; then place it in a dark environment at 4℃ for 2-5 days.

[0024] 3. After low-temperature treatment, cut off the flag leaf and stem of the young spikelet, disinfect it with alcohol, then rinse it with sterile distilled water to remove the surface alcohol; then soak it in sodium hypochlorite solution, and then rinse the young spikelet with sterile distilled water.

[0025] 4. In a sterile environment, use tweezers to peel off the flag leaf sheath of the young spikelet, take out the young spikelet, and then use scissors to cut off the spikelets at the top and bottom of the young spikelet, leaving the middle spikelet; take the two outermost florets of the retained spikelet, open the individual florets to expose the anthers inside, carefully pick out the anthers with tweezers and place them on the induction medium, then seal the culture dish and place it in a constant temperature and dark environment at 27±1℃ to induce callus culture.

[0026] In step one, young spikes from the early to mid-heading stage of hybrid F1 generation sheepgrass plants are selected.

[0027] In step one, the parents are either female gray-green type × male yellow type, female yellow-green type × male gray-green type, or female yellow type × male yellow-green type;

[0028] The induction medium in step four is MS medium containing 2.0 mg / L 2,4-dichlorophenoxyacetic acid (2,4-D) + 30 g / L sucrose + 7 g / L agar.

[0029] In this embodiment, the emergence of young spikelets accounting for 1 / 3 of the total spikelet length is the early heading stage of the sheepgrass plant, and the emergence of young spikelets accounting for 1 / 2 of the total spikelet length is the middle heading stage of the sheepgrass plant.

[0030] The preparation method of the induction medium in step four is as follows: Weigh 4.74g of MS powder and 30g of sucrose into a beaker using an analytical balance, add an appropriate amount of distilled water and stir with a glass rod until fully dissolved, then add 2.0mg / L of 2,4-D stock solution using a pipette; after the medium is brought to a final volume (1L), adjust the pH to 5.8, add 7g of agar powder, sterilize at 121℃ for 20min, and then dispense it into petri dishes while hot to prepare the induction medium described in this embodiment.

[0031] Specific Implementation Method Two: This implementation method differs from Specific Implementation Method One in that the young spikelets obtained in step one are inserted with the cut end facing down into a container filled with clean water for storage and transportation. All other steps and parameters are the same as in Specific Implementation Method One.

[0032] Specific Implementation Method Three: This implementation method differs from Specific Implementation Method One or Two in that, in step one, under sunny and stable weather conditions, the young spikelets are cut during the time period of 9:00-10:00 AM and / or 2:00-3:00 PM. All other steps and parameters are the same as in Specific Implementation Method One or Two.

[0033] Specific Implementation Method Four: This implementation method differs from Specific Implementation Methods One to Three in that, in step one, the young spikelet is cut at the second-to-last node of the Leymus chinensis plant. All other steps and parameters are the same as in Specific Implementation Methods One to Three.

[0034] Specific Implementation Method Five: This implementation method differs from Specific Implementation Methods One to Four in that the number of intermediate spikelets retained is 6 to 8. All other steps and parameters are the same as in Specific Implementation Methods One to Four.

[0035] Specific Implementation Method Six: This implementation method differs from Specific Implementation Methods One to Five in that, in step two, the young spikelets are wrapped with 2 to 3 layers of plastic wrap. All other steps and parameters are the same as in Specific Implementation Methods One to Five.

[0036] Example 1

[0037] This embodiment describes a method for culturing callus tissue from Leymus chinensis anthers:

[0038] 1. Select young spikelets from F1 generation hybrid Leymus chinensis plants with gray-green morphology as the female parent and yellow morphology as the male parent; insert the cut ends of the obtained young spikelets into a container filled with clean water for preservation and transportation; under sunny and stable weather conditions, cut the young spikelets between 9:00 and 10:00 am; the cut ends of the young spikelets should be located at the second-to-last node of the Leymus chinensis plant.

[0039] 2. Wrap the cut end of the young spike with a damp paper towel to keep it moist, and then wrap the entire spike with 2-3 layers of plastic wrap to prevent moisture loss; then place it in a dark environment at 4℃ for 2, 3, 4, 5 and 6 days respectively.

[0040] 3. After low-temperature treatment, cut off the flag leaf and stem of the young spikelet, disinfect it with 75% alcohol for 30 seconds in a clean bench, then rinse it twice with sterile distilled water to remove the surface alcohol; then soak it in 5% sodium hypochlorite solution for 6 minutes, and then rinse the young spikelet three times with sterile distilled water; place it in a petri dish with moist sterile filter paper for later use.

[0041] 4. In a sterile environment (clean bench), use tweezers to peel open the flag leaf sheath of the young spikelet, remove the young spikelet, and then use scissors to cut off the upper and lower spikelets, retaining 6-8 spikelets in the middle; take the two outermost florets of the retained spikelets, open each floret to expose the three anthers inside, carefully pick out the anthers with tweezers and place them on the induction medium, then seal the petri dish and place it in a constant temperature and dark environment at 27±1℃ to induce callus culture; after about 30 days of anther induction culture, the callus induction rate is calculated.

[0042] Callus induction rate = (Number of anthers producing callus / Total number of anthers) × 100%;

[0043] In step one, select young spikelets from the early to mid-heading stage of hybrid F1 generation *Leymus chinensis* plants. After removing the young spikelets, use scissors to cut off the immature spikelets at the top and bottom, retaining 6-8 spikelets in the middle. Take the two outermost florets from each spikelet, open each floret to expose the three anthers inside, carefully remove the anthers with tweezers, place them on the induction medium, then seal the petri dish with sealing film and place it in a constant temperature and dark environment at 27℃ to induce callus culture. The callus induction rate is calculated after approximately 30 days of anther induction culture. Callus induction rate = (Number of anthers producing callus / Total number of anthers) × 100%.

[0044] The induction medium in step four is MS medium containing 2.0 mg / L 2,4-dichlorophenoxyacetic acid (2,4-D) + 30 g / L sucrose + 7 g / L agar.

[0045] In this embodiment, the emergence of young spikelets accounting for 1 / 3 of the total spikelet length is the early heading stage of the sheepgrass plant, and the emergence of young spikelets accounting for 1 / 2 of the total spikelet length is the middle heading stage of the sheepgrass plant.

[0046] The preparation method of the induction medium in step four is as follows: Weigh 4.74g of MS powder and 30g of sucrose into a beaker using an analytical balance, add an appropriate amount of distilled water and stir with a glass rod until fully dissolved, then add 2.0mg / L of 2,4-D stock solution using a pipette; after the medium is brought to a final volume (1L), adjust the pH to 5.8, add 7g of agar powder, sterilize at 121℃ for 20min, and then dispense it into petri dishes while hot to prepare the induction medium described in this embodiment.

[0047] In this embodiment, the total number of anthers pretreated at low temperatures for 2, 3, 4, 5, and 6 days were 201, 265, 145, 166, and 122, respectively, corresponding to 11, 15, 5, 7, and 0 callus tissues induced. According to the formula for calculating the callus induction rate of anthers, the callus induction rates for 2, 3, 4, 5, and 6 days of low-temperature treatment were 5.5%, 5.7%, 3.4%, 4.2%, and 0, respectively. Therefore, low-temperature treatment for 2-5 days is beneficial for inducing more callus tissue, while treatment beyond 6 days makes it difficult to induce callus tissue. During the anther culture process, low-temperature treatment for 15 and 20 days was also attempted. A small amount of callus tissue was still produced, but it was not embryogenic and could not differentiate into seedlings. Furthermore, with excessively long low-temperature treatment times, the anthers of *Leymus chinensis* turned from green to yellow and quickly dried and browned during the induction culture process, failing to induce callus tissue.

[0048] Example 2

[0049] The difference between this embodiment and Embodiment 1 is that the young spikelets are treated at low temperature in an environment of 4℃ for 4 days; in step four, after the young spikelets are taken out in the ultra-clean workbench, the spikelets in the middle and lower part of the young spikelets are cut off with scissors, leaving only the upper 3 spikelets, and the anthers of the outermost two florets of each spikelet are taken for induction culture.

[0050] Example 3

[0051] The difference between this embodiment and embodiment 2 is that in step four, after taking out the young spikelet in the ultra-clean workbench, the middle and upper spikelets of the young spikelet are cut off with scissors, leaving only the lower 3 spikelets, and the anthers of the outermost two florets of each spikelet are taken for induction culture.

[0052] According to the statistical culture of the total number of anthers in Examples 1-3 (Example 1 is calculated based on 4 days of low temperature treatment in a 4℃ environment), the topmost and bottommost spikelets of the rachis usually only develop 1-2 florets between the early and middle stages of heading, and it is difficult for them to develop more than two florets. The number of anthers on the upper, middle and lower parts of the same spike is about 15, 40 and 15 respectively. The anthers near the top and bottom are shorter and develop later, making them easier to damage when picking them, thus reducing the callus induction rate.

[0053] Example 4

[0054] The difference between this embodiment and Embodiment 1 is that the plant was subjected to low-temperature treatment at 4°C for 4 days; and the young spikelets of F1 generation sheepgrass plants of the following types were selected: ♀ gray-green type × ♂ yellow-green type, ♀ yellow type × ♂ gray-green type, ♀ gray-green type × ♂ yellow type, ♀ yellow-green type × ♂ gray-green type, and ♀ yellow type × ♂ yellow-green type.

[0055] In the experiment, the highest callus induction rates of the F1 generation of female gray-green type × male yellow-green type, female yellow type × male gray-green type, female gray-green type × male yellow type, female yellow-green type × male gray-green type, and female yellow type × male yellow-green type were 3.6%, 22.2%, 25.9%, 27.9%, and 28.6%, respectively.

[0056] Statistical results from experiments showed that the highest callus induction rate was observed in the F1 generation of hybrids using the yellow-type Leymus chinensis as the female parent and the yellow-green type as the male parent, making it suitable as material for inducing callus formation in Leymus chinensis anthers. When the gray-green type was used as the female parent and the yellow-type Leymus chinensis as the male parent, the callus induction rate in the F1 generation reached 25.9%. However, when the yellow-green type was used as the male parent, the callus induction rate in the F1 generation was only 3.6%. This indicates that the callus induction rate of Leymus chinensis is highly dependent on the ecotype, and selecting a suitable ecotype can significantly improve the callus induction rate.

Claims

1. A method for improving the callus induction rate in Leymus chinensis anther culture, characterized in that, This method is performed in the following steps: I. Select young spikelets from F1 generation hybrids of different ecotypes of Leymus chinensis; 2. Wrap the cut end of the young spike with a damp paper towel to keep it moist, then wrap the entire spike with plastic wrap to prevent moisture loss. After that, place it in a dark environment at 4℃ for 2 to 5 days.

3. After low-temperature treatment, cut off the flag leaf and stem of the young spikelet, disinfect it with alcohol, then rinse it with sterile distilled water to remove the surface alcohol, then soak it in sodium hypochlorite solution, and then rinse the young spikelet with sterile distilled water.

4. In a sterile environment, use tweezers to peel off the flag leaf sheath of the young spikelet, take out the young spikelet, and then use scissors to cut off the spikelets at the top and bottom of the young spikelet, leaving the middle spikelet; take the two outermost florets of the retained spikelet, open the individual florets to expose the anthers inside, carefully pick out the anthers with tweezers and place them on the induction medium, then seal the culture dish and place it in a constant temperature and dark environment at 27±1℃ to induce callus culture. In step one, young spikes from the early to mid-heading stage of hybrid F1 generation sheepgrass plants are selected. In step one, the parents are either female gray-green type × male yellow type, female yellow-green type × male gray-green type, or female yellow type × male yellow-green type; The induction medium in step four is MS medium containing 2.0 mg / L 2,4-dichlorophenoxyacetic acid, 30 g / L sucrose, and 7 g / L agar.

2. The method for improving the callus induction rate in Leymus chinensis anther culture according to claim 1, characterized in that, The young spikelets obtained in step one are inserted with the cut end facing down into a container filled with clean water for storage and transportation.

3. The method for improving the callus induction rate in Leymus chinensis anther culture according to claim 1 or 2, characterized in that, In step one, under sunny and stable weather conditions, select the time period of 9:00-10:00 am and / or 2:00-3:00 pm to cut the young spikelets.

4. The method for improving the callus induction rate in Leymus chinensis anther culture according to claim 3, characterized in that, In step one, the cut of the young spikelet is located at the second node from the bottom of the sheepgrass plant.

5. The method for improving the callus induction rate in Leymus chinensis anther culture according to claim 4, characterized in that, The number of intermediate spikelets retained is 6 to 8.