Exciton peptide LilyPep1 and application thereof in promoting tissue culture and rapid propagation of lilium brownii
By adding the elicitor peptide LilyPep1 during lily tissue culture, the problems of declining lily quality and varietal degeneration were solved, achieving efficient and high-quality propagation of lily through rapid tissue culture, promoting lily growth and stress resistance, and meeting production needs.
Patent Information
- Application Number
- CN202511080576.8
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-08-04
- Publication Date
- 2025-11-04
AI Technical Summary
In existing technologies, lily production suffers from quality decline and varietal degradation, and the application of tissue culture technology in the rapid propagation of lily tissue culture has not yet addressed the impact of elicitor peptides.
Adding specific concentrations of lily elicitor peptide LilyPep1 to the culture medium at each stage of lily development, including primary culture, subculture proliferation, and rooting culture, and optimizing the culture medium composition and treatment steps promotes rapid propagation of lily tissue culture.
It significantly improved the germination rate, growth rate, and rooting rate of lily tissue culture seedlings, shortened the culture cycle, enhanced the physiological activity and stress resistance of tissue culture seedlings, maintained the excellent characteristics of lily varieties, and met production needs.
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Figure CN120887965A_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the field of plant tissue culture technology, specifically to the elicitor peptide LilyPep1 and its application in promoting rapid propagation of lily tissue culture. Background Technology
[0002] Lanzhou Lily ( Lilium davidii var.unicolor) It belongs to the genus *Lilium* in the family Liliaceae, a subclass of monocotyledons. It is a perennial bulbous herbaceous plant and the only edible sweet lily variety in China. Lilies contain various active ingredients such as steroidal saponins, phenols, alkaloids, and polysaccharides, and have the effects of nourishing yin and moistening the lungs, calming the mind and soothing the nerves, relieving fever and promoting diuresis, relieving cough and asthma, and regulating the stomach and spleen. Currently, lily production is mostly carried out by farmers themselves, resulting in a serious decline in quality and varietal degeneration.
[0003] Tissue culture technology boasts advantages such as high efficiency, virus removal, and uniformity, meeting the large-scale needs of production and serving as an effective way to address the aforementioned varietal degeneration phenomena. Existing lily research has explored the impact of different explant selections on tissue culture, methods for disinfecting lily explants, and the optimal culture medium formulations for lilies at different cultivation stages. However, in recent years, the influence of elicitor peptides on the rapid propagation of lily tissues has not been identified. Summary of the Invention
[0004] To address the aforementioned technical problems, this invention provides the elicitor peptide LilyPep1 and its application in promoting rapid propagation of lily tissue culture. The promoting effect of Lily elicitor peptide LilyPep1 in rapid propagation of lily tissue culture was investigated by adding a certain concentration of Lily elicitor peptide LilyPep1 to the culture medium at various stages of lily development, as detailed below: A lily elicitor peptide, LilyPep1, wherein the amino acid sequence of LilyPep1 is shown in SEQ ID NO:2 and the nucleotide sequence of LilyPep1 is shown in SEQ ID NO:1.
[0005] On the other hand, the present invention provides the application of the lily elicitor peptide LilyPep1 to promote the growth of lily plants in tissue culture, comprising the following steps: S1. Explant disinfection: Take lily scales, disinfect them, and then rinse them with sterile water. S2. Explant treatment: Place the sterilized lily scales in a culture dish containing primary liquid culture medium, cut them into pieces, then vacuum for 30-60 min, let stand for 1.0-2.0 h, and wipe dry. S3, Primary culture: Lily scales are cultured in the dark in primary liquid culture medium for 3-4 weeks to obtain primary shoots; S4. Subculture and Proliferation: Transfer the primary shoots to the subculture medium and culture them for 50-60 days to obtain proliferating shoots; S5, rooting culture: the proliferation buds are transferred to the rooting culture medium and cultured for 20-30 days to obtain lily tissue culture seedlings.
[0006] The growth conditions of each stage are shown in Figure 7 .
[0007] Further, in the step S1, the disinfection method is: cleaning with food-grade vegetable detergent and rinsing with running water for 0.5h-1h, first disinfecting with 75% alcohol for 30s, then disinfecting with 1% NaClO for 8min, and then rinsing with sterile water for 2-3 times.
[0008] Further, in the step S2, the primary liquid culture medium is: MS medium+1 mg / L 6-BA+0.5 mg / L NAA and 10 nM LilyPep1.
[0009] Further, in the step S4, the subculture medium is: MS medium+0.5 mg / L 6-BA+0.2 mg / L NAA+10 nM LilyPep1.
[0010] Further, in the step S5, the rooting culture medium is: 1 / 2 MS medium+0.5 mg / L IBA, 0.8 mg / L NAA and 10 nM LilyPep1.
[0011] Further, in the step S2, the culture temperature is 25±2℃, and the humidity is 40%-80%.
[0012] Further, in the step S4, the culture temperature is 25±2℃, and the humidity is 40%-60%.
[0013] Further, in the step S5, the culture temperature is 25±2℃, and the humidity is 40%-60%.
[0014] Compared with the prior art, the beneficial effects of the present application are: 1、The elicitor peptide LilyPep1 provided by the present application is applied in lily tissue culture, in the primary culture, the lily explant with 10 nM lily elicitor peptide LilyPep1 has a sprouting rate of 92.59%, which is 17.59% higher than the sprouting rate of 75% of the control without lily elicitor peptide LilyPep1; in the subculture, the average length of the plant height reaches 3.27 cm, which is 48.64% higher than the average length of 2.2 cm of 0 nM; in the rooting culture, the rooting rate reaches 83.33% after adding the lily elicitor peptide LilyPep1, which is 25.33% higher than the rooting rate of 58% of the control without lily elicitor peptide LilyPep1.
[0015] 2、By adding specific concentration of elicitor peptide LilyPep1 in each stage of lily medium, the time cycle of lily tissue culture in each stage is effectively shortened, and the elicitor peptide LilyPep1 promotes cell division and differentiation, accelerates the formation of buds and roots, so that the efficiency of lily tissue culture and rapid propagation is greatly improved, and a large number of high-quality lily tissue culture seedlings can be obtained faster to meet the large demand of lily seedlings in production.
[0016] 3、The lily elicitor peptide LilyPep1 used in the present application not only promotes the growth of lily, but also enhances the physiological activity and stress resistance of the tissue culture seedlings, compared with the conventional culture method without adding LilyPep1, the lily tissue culture seedlings obtained after adding LilyPep1 grow more robustly, the root system is developed, the leaf is emerald green and lustrous, and the resistance to diseases and pests is enhanced, effectively reducing the management cost and loss in the subsequent cultivation process, and improving the planting benefit of lily.
[0017] 4、In existing lily production, the quality is seriously degraded and the variety is seriously degenerated due to self-production and self-propagation, the present application uses tissue culture technology combined with the effect of lily elicitor peptide LilyPep1 to maintain the excellent characteristics of lily varieties and realize rapid and high-quality propagation of lily, providing an effective technical means to solve the problem of lily variety degeneration, which helps to maintain and improve the market competitiveness and brand value of Lanzhou lily.
[0018] 5、The present application first applies the elicitor peptide LilyPep1 to lily tissue culture, creating a brand-new technical approach to promote lily tissue culture and rapid propagation, providing a new idea and method for the field of plant tissue culture, and enriching the research and application scope of plant growth regulators. BRIEF DESCRIPTION OF DRAWINGS
[0019] Figure 1 Purity detection of lily elicitor peptide LilyPep1; Figure 2 Molecular weight verification of lily elicitor peptide LilyPep1; Figure 3 Bud emergence results of primary culture medium optimized by orthogonal design; Figure 4 Results of lily elicitor peptide LilyPep1 promoting bud induction in primary culture; Figure 5 Comparison of results of lily elicitor peptide LilyPep1 promoting proliferation culture; Figure 6 Results of lily elicitor peptide LilyPep1 promoting lily rooting culture; Figure 7To investigate the effect of the peptide LilyPep1 in promoting rapid propagation of lily tissue culture. Detailed Implementation
[0020] Example 1: Isolation and identification of the lily elicitor peptide LilyPep1 sequence Sequence alignment was performed using the amino acid sequence of the Arabidopsis homology gene AtPep1 (AT5G64900.1). LilyPep1 was isolated and identified from the Lanzhou lily genome. Upstream primer LilyPep1-F and downstream primer LilyPep1-R (LilyPep1-F: SEQ ID NO:3; LilyPep1-R: SEQ ID NO:4) were designed. The corresponding sequence of LilyPep1 was amplified by PCR using alfalfa cDNA as a template. The high-fidelity enzyme 2xPhanta FlashMaster Mix (Catalog No.: P520) from Novizan Biosciences was selected to ensure the fidelity of the amplified product. After PCR electrophoresis, the PCR product was sent to Sangon Biotech (Shanghai) Co., Ltd. for sequencing. The sequencing result of the PCR product is shown in SEQ ID NO:1, which is the sequence of LilyPep1. The amino acid sequence of LilyPep1 is shown in SEQ ID NO:2.
[0021] Example 2 Synthesis of Lily Pep1, a Lily Evotron Peptide The lily elicitor peptide LilyPep1 was synthesized using Fmoc solid-phase synthesis technology and an Applied Biosystems 431 synthesizer. The synthesis consisted of the following cycles: Deprotection: Fmoc-protected columns and monomers must have their amino protecting groups removed using an alkaline solvent (piperidine); Activation and cross-linking: The carboxyl group of the next amino acid is activated by an activator, and the activated monomer reacts with the free amino group to cross-link and form a peptide bond. In this step, a large amount of ultra-concentrated reagent is used to drive the reaction to completion.
[0022] Cycle: These two steps are repeated until the synthesis is complete; Elution and Deprotection: The lily elicitor peptide LilyPep1 was eluted from the column, and its protecting group was eluted and deprotected by a deprotecting agent (TFA). The synthesized peptide was purified and detected by C18 reversed-phase high-performance liquid chromatography. Figure 1 ), and molecular weight verification was performed using a Finnigan LCQ mass spectrometer via direct sample injection. Figure 2 ).
[0023] Example 3: Optimization of explant treatment Disinfection of explants: Take healthy and full lily scales as explants, wash them with food-grade vegetable detergent and rinse them with running water for 0.5-1 h, first disinfect them with 75% alcohol for 30 s, then disinfect them with 1% NaClO for 8 min, and finally rinse them with sterile water for 2-3 times.
[0024] Treatment of explants: Place the above-mentioned disinfected explants on sterile filter paper to absorb surface moisture, place them in a culture dish containing primary liquid medium, cut them into small pieces of about 1 cm*1 cm, then vacuum for 30-60 min, stand for 1.0-2.0 h, dry the treated explants, and inoculate them into primary solid medium. Incubate in the dark in an incubator at a temperature of (25±2)℃ and a humidity of 40%-80%, and count the induction rate after 3-4 weeks.
[0025] Example 4 Optimization of optimal primary culture medium Take MS medium as the basis, select two hormones that have a greater impact on lily tissue culture: plant growth regulator 6-benzylaminopurine (6-BA) and naphthalene acetic acid (NAA), and use orthogonal test design (two factors, three levels) to optimize the addition amount. Each factor is set to two levels, 6-BA concentration is 1.0 mg / L and 3.0 mg / L, and NAA concentration is 0.1 mg / L and 0.5 mg / L (Table 1). Each test is repeated three times, and after 21 days of primary culture, the absolute budding rate (number of budding explants / total number of explants x 100%) and the relative budding rate (total number of buds / number of budding explants) are counted. The results show (Table 1, Figure 3 ), the absolute budding rates of experimental groups one and two are both 76.67%, which is significantly higher than 53.33% and 46.67% of experimental groups three and four, an increase of 23.34% and 30%, indicating that experimental groups one and two are better.
[0026] Table 1 Budding statistics of primary culture
[0027] Example 5 LiliPep promotes bud induction in primary culture On the basis of the above-mentioned optimal primary culture medium, add 0 nM, 1 nM and 10 nM of lily elicitor peptide LilyPep1, respectively, and use no addition of lily elicitor peptide LilyPep1 as a control. After 30 days of culture, it is found that the lily explant budding rate of 10 nM lily elicitor peptide LilyPep1 is 92.59%, which is significantly higher than the 75% budding rate of the control without lily elicitor peptide LilyPep1, an increase of 17.59% (Table 2, Figure 4); therefore, the optimization result of the initial culture medium lily elicitor peptide LilyPep1 is experiment group three: MS + 1 mg / L 6-BA + 0.5 mg / L NAA + 10 nM lily elicitor peptide LilyPep1.
[0028] Table 2. Lily elicitor peptide LilyPep1 promotes the initial culture of buds
[0029] Example 6. Application of lily elicitor peptide LiliPep to promote subculture proliferation On the basis of subculture proliferation medium MS medium + 0.5 mg / L 6-BA + 0.2 mg / L NAA, 0 nM, 1 nM and 10 nM lily elicitor peptide LilyPep1 were added respectively (Table 3), and no lily elicitor peptide LilyPep1 was added as a control, and cultured for 60 days. The results are shown in Table 3, Figure 5 The proliferation coefficient of 10 nM lily elicitor peptide LilyPep1 was 3.25, which was significantly improved by 80.56% compared with 1.8 of no lily elicitor peptide LilyPep1; the average length of 10 nM lily elicitor peptide LilyPep1 was 3.27 cm, which was significantly improved by 48.64% compared with 2.2 cm of 0 nM.
[0030] Table 3. Lily elicitor peptide LilyPep1 promotes proliferation culture proliferation results
[0031] Example 7. Application of lily elicitor peptide LiliPep1 to promote rooting On the basis of rooting culture medium 1 / 2 MS medium + 0.5 mg / L IBA + 0.8 mg / L NAA, 0 nM, 1 nM and 10 nM lily elicitor peptide LilyPep1 were added respectively (Table 4), and no lily elicitor peptide LilyPep1 was added as a control, and cultured for 30 days. The results are shown in Table 4, Figure 6 The rooting rates of 5 nM and 10 nM lily elicitor peptide LilyPep1 were 75% and 83.33% respectively, which were significantly improved by 17% and 25.33% respectively compared with the rooting rate of 58% of no lily elicitor peptide LilyPep1, indicating that lily elicitor peptide LilyPep1 significantly promotes lily rooting.
[0032] Table 4. Test design and results of lily elicitor peptide LilyPep1 promoting rooting culture
Claims
1. A lily elicitor peptide, LilyPep1, characterized in that, The amino acid sequence of LilyPep1 is shown in SEQ ID NO:2, and the nucleotide sequence of LilyPep1 is shown in SEQ ID NO:
1.
2. The application of the lily elicitor peptide LilyPep1 as described in claim 1, characterized in that, Used to promote the growth of lily plants in tissue culture.
3. The application of the lily elicitor peptide LilyPep1 as described in claim 2, characterized in that, Includes the following steps: S1. Explant disinfection: Take lily scales, disinfect them, and then rinse them with sterile water. S2. Explant treatment: Place the sterilized lily scales in a culture dish containing primary liquid culture medium, cut them into pieces, then vacuum for 30-60 min, let stand for 1.0-2.0 h, and wipe dry. S3, Primary culture: Lily scales are cultured in the dark in primary liquid culture medium for 3-4 weeks to obtain primary shoots; S4. Subculture and Proliferation: Transfer the primary shoots to the subculture medium and culture them for 50-60 days to obtain proliferating shoots; S5. Rooting culture: Transfer the proliferating buds to the rooting medium and culture for 20-30 days to obtain lily tissue culture seedlings.
4. The application of the lily elicitor peptide LilyPep1 as described in claim 3, characterized in that, In step S1, the disinfection method is as follows: wash with food-grade vegetable detergent and rinse with running water for 0.5-1 hour, disinfect with 75% alcohol for 30 seconds, then disinfect with 1% NaClO for 8 minutes, and then rinse with sterile water 2-3 times.
5. The application of the lily elicitor peptide LilyPep1 as described in claim 3, characterized in that, In step S2, the initial liquid culture medium is: MS medium + 1 mg / L 6-BA + 0.5 mg / L NAA and 10 nM LilyPep1.
6. The application of the lily elicitor peptide LilyPep1 as described in claim 3, characterized in that, In step S4, the subculture medium is: MS medium + 0.5 mg / L 6-BA + 0.2 mg / L NAA + 10 nM LilyPep1.
7. The application of the lily elicitor peptide LilyPep1 as described in claim 3, characterized in that, In step S5, the rooting medium is: 1 / 2 MS medium + 0.5 mg / L IBA + 0.8 mg / L NAA + 10 nM LilyPep1.
8. The application of the lily elicitor peptide LilyPep1 as described in claim 3, characterized in that, In step S2, the culture temperature is 25±2℃ and the humidity is 40%-80%.
9. The application of the lily elicitor peptide LilyPep1 as described in claim 3, characterized in that, In step S4, the culture temperature is 25±2℃ and the humidity is 40%-60%.
10. The application of the lily elicitor peptide LilyPep1 as described in claim 3, characterized in that, In step S5, the culture temperature is 25±2℃ and the humidity is 40%-60%.