Young ear continuous induction method for tissue culture of pennisetum americanum
By planting in restricted nutrient soil and combining environmental control with regular stem segment collection, the problem of the unsustainable supply of young spikelets of *Pennisetum alopecuroides* was solved, enabling efficient and large-scale acquisition of young spikelets and promoting gene function verification.
Patent Information
- Application Number
- CN202610107569.0
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2026-01-27
- Publication Date
- 2026-03-17
AI Technical Summary
The lack of existing methods for cultivating young spikelets of Napier grass that can be continuously produced for tissue culture makes it difficult to quickly obtain young spikelets at the optimal harvesting period in large quantities, thus limiting the progress of gene function verification.
By planting American foxtail grass seedlings in restricted nutrient soil, combined with environmental control and artificial intervention, including restricting root nutrient space, transferring to large containers to provide sufficient nutrients, and stimulating lateral bud germination by regularly collecting stem segments, a continuous young spikelet production cycle is formed.
This method enables the continuous and efficient production of young spikelets of *Pennisetum purpureus*, shortens the time required to determine the developmental stage, provides a large amount of high-quality embryogenic callus tissue for transgenic verification, and promotes gene function research.
Smart Images
Figure CN121667104A_ABST
Abstract
Description
Technical Field
[0002] This invention relates to the fields of plant physiology and molecular biology, specifically to a method for the continuous induction of young spikelets in tissue culture of *Pennisetum purpureus*. Background Technology
[0003] American foxtail grass (Pennisetum glaucum (L.) R. Br., syn. Cenchrus americanus (L.) Morrone) is an annual C4 plant belonging to the genus Pennisetum in the Poaceae family. It is widely used as a forage grass, primarily for the aquaculture of herbivorous poultry and freshwater fish. Because American foxtail grass thrives in high temperatures and grows rapidly in summer, its heat tolerance mechanisms have attracted considerable attention. Currently, the whole genome, graphical pan-genome, and single-cell sequencing of American foxtail grass have been published, providing a solid omics foundation for studying the molecular mechanisms of heat tolerance. However, the lack of a genetic transformation system limits the functional research of heat tolerance genes. In the field, American foxtail grass can reach a height of 4-5 meters. After entering the reproductive stage, the apical meristem differentiates into young spikelets. These young spikelets are the best explants for inducing embryogenic callus in tissue culture, and are important tissue materials for obtaining transgenic American foxtail grass plants and verifying gene function. After the flowering process of *Napierus spp.* begins, when the flag leaf tip is just emerging but has not yet protruded from the leaf cushion, the apical meristem has completed spike differentiation, but the young spike is still enclosed within the stem. This is the critical period for obtaining young spikes for tissue culture, but it is usually difficult to determine quickly. Therefore, rapid and continuous collection of a sufficient number of young spikes at the optimal sampling period is a key step in conducting *Napierus spp.* tissue culture for gene function verification. Although young spikes are ideal explants for inducing embryogenic callus and obtaining transgenic *Napierus spp.*, one of the key bottlenecks in the entire technical system is the difficulty in ensuring a continuous supply of young spikes throughout the growing season.
[0004] A search revealed that, to date, there are no reports in the existing technology of cultivating young spikelets of *Pennisetum purpureus* that can be continuously produced for tissue culture. Summary of the Invention
[0005] The purpose of this invention is to provide a method for the continuous induction of young spikelets for tissue culture of *Pennisetum purpureum*, which can be used to obtain the best young spikelet explants for tissue culture and contribute to the study of gene function in *Pennisetum purpureum*.
[0006] The objective of this invention is achieved as follows: This method includes the following steps: ① Planting American foxtail grass seedlings in limited nutrient soil: When the average daily temperature is stable at 20-25℃, transplant the sprouted seedlings of American foxtail grass into small square pots (such as 5cm×5cm×5cm). The pot contains limited nutrient soil. Place this potted plant outdoors in a natural environment to grow. The root restriction at this stage helps to control the plant height and induce the plant to enter the reproductive growth stage. ②Prepare a large container filled with sufficient nutrient soil: Prepare a large container (such as a turnover box), line the inside with a water-permeable and leak-proof cloth, and fill it with about two-thirds of its volume of nutrient soil. Water it thoroughly before use. ③ After the American foxtail grass enters its reproductive growth period, place the small square pot with seedlings into a large container with sufficient nutrients: When the plant has grown 6-9 stem nodes and some plants have begun to produce spikelets, you can check whether the meristematic tissue has differentiated into young spikelets by checking whether there are leaf tips at the hollow top of the stem segment. This will help determine whether the plant that has not yet produced spikelets has entered the reproductive growth stage. After entering the reproductive growth stage, after obtaining the main stem segment that is producing young spikelets, place the entire small square pot with the seedling into the prepared large container. The roots of the American foxtail grass will penetrate through the drainage holes at the bottom of the small pot and extend into the nutrient soil in the large container to absorb nutrients. After that, careful management is required. Fertilize once a week to ensure nutrient supply and water once every other day to ensure sufficient moisture. ④ Be sure to collect stem segments every 3-4 days: After the initial collection of stem segments from the young spikelets, newly emerging lateral bud stem segments are harvested every 3-4 days. Thinner lateral bud stem segments can be harvested when they have 2-4 fully expanded leaves, while thicker lateral bud stem segments can be harvested when they have 4-8 fully expanded leaves. This harvesting frequency can, on the one hand, obtain high-quality young spikelets that induce embryogenic callus; on the other hand, by removing apical dominance, it continuously stimulates the germination of a new round of lateral buds and the differentiation of young spikelets, forming a stable young spikelet production cycle that can last for 3 months.
[0007] This method has the following advantages and positive effects: ① It can control the plant height of American foxtail grass, making it easier to harvest young spikelets; ② It can produce stem segments that develop young spikelets for about 3 consecutive months after a single planting; ③ It can achieve rapid and large-scale collection of stem segments that are developing young spikelets in a single operation, thereby significantly shortening the time required to determine the developmental stage of the young spikelets, obtaining young spikelets for the production of embryogenic callus, facilitating the acquisition of transgenic materials, and accelerating the verification of the gene function of *Pennisetum purpureum*. Attached Figure Description
[0008] Figure 1 This is a flowchart illustrating the method. ① Seedling raising; ② Entering reproductive growth; ③ Prepare fresh seedling substrate; ④ Place the seedlings in the frame and fertilize weekly; ⑤ Maintaining a fixed collection frequency ensures the continuous production of young spikelets; ⑥ Collect young spikelet branches; ⑦ Remove the outer leaves from the branches; ⑧ Disinfect the surface of young spikelet branches; ⑨ Remove the external tissues of the young spikelet.
[0009] Figure 2 The image shows stem segments formed from lateral buds of *Pennisetum purpureum* and the developing young spikelets. A—The stem segments formed by the lateral buds of *Pennisetum purpureum* after the main stem segment has entered the reproductive growth stage; B—The lateral bud develops into a stem segment with 4 fully expanded leaves, and the tender young spikelet that can be used for tissue culture is shown after the leaves are peeled off. ①②③④ represent the fully expanded leaves or the stem node where the leaves are attached. Detailed Implementation
[0010] The following is a detailed description with reference to the accompanying drawings and embodiments: like Figure 1 The process of this method: ① Planting American foxtail grass seedlings in small square pots Once the midday temperature reaches 20-25℃, plant the sprouting American foxtail grass seedlings in small square pots (e.g., 5cm×5cm×5cm) and place them outdoors in a natural environment to grow.
[0011] ②American foxtail grass enters its reproductive growth stage When the plant has grown 6-9 stem nodes and some plants have begun to produce spikelets, you can check whether the meristematic tissue has differentiated into young spikelets by checking whether there are leaf tips in the hollow part of the stem segment. This will help determine whether the plant that has not yet produced spikelets has entered the reproductive growth stage.
[0012] ③Prepare turnover baskets filled with nutrient soil Prepare a transfer basket, lined with a permeable cloth to prevent soil leakage, fill it with 2 / 3 nutrient soil, and water it thoroughly before use.
[0013] ④ Place the plant, along with its small pot, into a transfer basket filled with nutrient soil as it enters reproductive growth. After entering the reproductive growth period, obtain the main stem segment that is bearing young spikelets, and place the small square pot with the seedling into the prepared turnover basket; the roots of the American foxtail grass will penetrate the bottom of the small pot and extend into the nutrient soil in the turnover basket to absorb nutrients; thereafter, it is necessary to carry out careful management, fertilize once a week to ensure nutrient supply, and water once every other day to ensure sufficient moisture.
[0014] ⑤ Maintain a fixed collection frequency of 3-4 days to ensure continuous production of young spikelets. After the first collection of stem segments that have developed young spikelets, newly emerging lateral bud stem segments are harvested every 3-4 days. This harvesting frequency can obtain high-quality young spikelets with induced embryogenic callus tissue. On the other hand, by removing apical dominance, it can continuously stimulate the germination of a new round of lateral buds and the differentiation of young spikelets, forming a stable production cycle of young spikelets that can last for 3 months.
[0015] ⑥ Collected young spikelet branches (see...) Figure 2 ) Collect branches when the thin lateral bud stem segments have 2-4 fully unfolded leaves; collect branches when the thick lateral bud stem segments have 4-8 fully unfolded leaves (some thick stem segments from individual lateral buds require more leaves before collecting branches; harvest promptly when the top is hollow and there are no leaf tips). ⑦ Remove the outer leaves from the branches Use scissors to cut off the leaves on the outside of the branch, leaving only the stem.
[0016] ⑧ Disinfect the outer surface of the young spikelet branches and remove the outer layer of tissue covering the young spikelet. Disinfect the stem surface by wiping it with a cotton ball soaked in 70% alcohol.
[0017] 9. Remove all tissues surrounding the young spikelet to obtain a sterile young spikelet. After wiping the stem surface with a cotton ball soaked in 70% alcohol in a clean bench, carefully peel off the outer tissue covering the young spikelet to obtain sterile young spikelet tissue.
[0018] Working principle: This invention provides a cultivation method for enabling American foxtail grass to continuously produce young spikelets. Its working principle combines environmental control, artificial intervention, and plant growth and development mechanisms. ① Controlling height and promoting flowering: In the early stage, the root system's nutrient space is restricted, which effectively dwarfs the plant and promotes it to enter the reproductive growth period; ② Sufficient fertilizer to promote bud growth: During the reproductive period, the plant is transferred to a large container and fertility is ensured. The activated flowering signals maintain the differentiation of lateral buds into young spikelets; ③ Harvesting promotes the cycle: By harvesting regularly every 3-4 days, new buds are continuously stimulated to grow, forming a virtuous cycle of "harvesting-budding".
[0019] The purpose of collecting young spikelets: In the field, *Pennisetum alopecuroides* can reach a height of 4-5 meters. After entering the reproductive stage, its apical meristem differentiates into young spikelets. These young spikelets are the optimal explants for inducing embryogenic callus in *Pennisetum alopecuroides* tissue culture, and are crucial tissue materials for obtaining transgenic *Pennisetum alopecuroides* plants and verifying gene function. After *Pennisetum alopecuroides* initiates its flowering program, when the plant's external morphology shows the flag leaf tip just emerging but not yet protruding from the leaf pulvinus, the apical meristem has completed spikelet differentiation, but the young spikelet is still enclosed within the stem. This is the critical period for obtaining young spikelets for tissue culture, but it is usually difficult to determine quickly. Therefore, rapidly collecting and continuously obtaining a sufficient number of young spikelets at the optimal collection period is a key step in conducting *Pennisetum alopecuroides* tissue culture for gene function verification.
[0020] The innovativeness of this invention: This invention is based on four findings from two years of inducing young spikelets in *Pennisetum purpureum*. It innovatively combines environmental control, artificial intervention, and molecular mechanisms of plant growth and development, and explores their application in practice, ultimately obtaining a cultivation method for *Pennisetum purpureum* to produce young spikelets continuously and efficiently. ① It was found that when American foxtail grass seedlings are planted in limited nutrient soil, they are shorter and weaker, but they will flower faster and make it easier to collect young spikelets.
[0021] ② It was found that after the American foxtail grass entered the reproductive growth stage, after removing the main stem that nurtures the young spikelets, the apical meristem of the lateral branches generated by the lateral buds will also quickly enter the reproductive growth stage, that is, the entire plant will irreversibly start the flowering program.
[0022] ③ It was discovered that the lateral buds of American foxtail grass have a strong regenerative ability under sufficient nutrient supply. Once the flowering process is initiated, new lateral branches that will produce young spikelets can be continuously generated by taking lateral branches.
[0023] ④ Interactions were found between summer light and temperature conditions, nutrient elements, the regeneration of lateral buds in *Pennisetum alopecuroides*, and the flowering program regulation mechanism. Experiments revealed that after initiating the flowering mechanism, it takes 3-4 days for lateral buds to grow into young spikelets suitable for tissue culture. However, robust lateral branches take longer to form young spikelets suitable for tissue culture, while smaller lateral branches take shorter times. The final standard for rapid batch collection was determined as follows: stem segments must be collected every 3-4 days. Thinner lateral bud stem segments with 2-4 fully expanded leaves can be collected; thicker lateral bud stem segments with 4-8 fully expanded leaves can be collected. Young spikelets meeting the callus induction requirements were selected for the experiment.
Claims
1. A method for continuous induction of young spikes for tissue culture of Puccinellia distans, characterized by: ① Puccinellia distans seedlings are planted in limited nutrient soil: When the daily mean temperature stabilizes at 20-25℃, Puccinellia distans germination seedlings are planted in small square pots with limited nutrient soil, and the pots are placed in the outdoor natural environment for growth. The root system restriction in this stage helps to control plant height and induce the plant to enter the reproductive growth stage; ② Prepare a large container filled with sufficient nutrient soil: Prepare a large container, line the inside with water-permeable leak-proof cloth, and fill about two-thirds of the volume with nutrient soil. Pour water thoroughly before use; ③ After Puccinellia distans enters the reproductive growth stage, place the small square pots with seedlings in the large container with sufficient nutrients: When the plant grows 6-9 stem nodes and individual plants are observed to begin to sprout, check whether the meristematic tissue has differentiated into young spikes by checking whether there are leaf tips in the hollow at the top of the stem section to determine whether the un-sprouting plants have entered the reproductive growth stage. After entering the reproductive growth stage, obtain the main stem section with young spikes, and then place the entire small square pot with seedlings into the prepared large container. The root system of Puccinellia distans will extend through the water leakage hole at the bottom of the small pot and into the nutrient soil in the large container to absorb nutrients. Thereafter, fine management is required, with fertilization once a week to ensure nutrient supply and watering once every other day to ensure adequate water supply; ④ Collect stem sections every 3-4 days: After the first collection of stem sections with young spikes, collect new sprouted lateral bud stem sections every 3-4 days. Thin lateral bud stem sections can be collected when the fully expanded leaves are 2-4 pieces, and thick lateral bud stem sections can be collected when the fully expanded leaves are 4-8 pieces. This collection frequency can obtain high-quality young spikes induced by embryonic callus, and on the other hand, by removing the apical dominance, it continuously stimulates the germination of a new round of lateral buds and the differentiation of young spikes, forming a 3-month sustainable young spike stable production cycle.