Method for inducing rapid rooting of lily bulbils and use thereof

Abstract

The application provides a method for inducing rapid rooting of lily bulbils and application thereof, and the lily bulbil (diameter>1.0cm) is used as material, the outer scale is peeled off after surface disinfection and low-temperature pretreatment, the peeled-off outer scale is placed in sterile sawdust with humidity of 15%-25% and cultured at 26 DEG C in dark, and the lily bulbil can be rooted. Compared with a control, the lily bulbil obtained by the method has developed root system, early root system germination, and the number and length of roots are obviously increased, the low-temperature pretreatment time is short, the culture period is short, and a large amount of lily root system can be rapidly obtained in about 10 weeks, and the lily root system can be applied to preparation of medicines, foods and cosmetics resisting melanin synthesis and inflammation reaction, and the method can also realize rapid propagation of lily and provide a new selection, and has important significance. The material is easy to obtain, the method is non-tissue culture, simple and convenient to operate, and can be produced on a large scale, and has good application prospect.

Description

Technical Field

[0001] This invention belongs to the technical field of lily cultivation, specifically relating to a method for inducing rapid rooting of lily bulbils and its application. Background Technology

[0002] Lilium lancifolium Thunb., also known as Yixing lily, medicinal lily, tiger skin lily, etc., is a perennial bulbous herbaceous plant belonging to the genus Lilium in the family Liliaceae. It is cultivated in most parts of my country. The bulb of Lilium lancifolium is a valuable traditional Chinese medicine with the effects of nourishing, strengthening, relieving cough, and expectorating phlegm. It has a good therapeutic effect on pulmonary tuberculosis and chronic bronchitis. Furthermore, studies have found that the methanol extract of the root of Lilium lancifolium has a significant anti-inflammatory effect (Kwon OK, Lee MY, Yuk JE, Oh SR, Chin YW, Lee HK, Ahn KS. Anti-inflammatory effects of methanol extracts of the root of Lilium lancifolium on LPS-stimulated Raw264.7 cells. J Ethnopharmacol. 2010, 130(1):28-34.). A research paper published by Korean scholars in the international journal *Plants* (Park S, Han N, Lee J, Lee JN, An S, Bae S. Anti-Melanogenic Effects of Lilium lancifolium Root Extract via Downregulation of PKA / CREB and MAPK / CREB Signaling Pathways in B16F10 Cells. *Plants* (Basel). 2023, 12(21): 3666.) reveals the mechanism by which *Lilium lancifolium* root extract inhibits melanin synthesis. Therefore, developing the medicinal value of its roots can not only serve as a novel, naturally derived therapeutic ingredient to improve pigmentation and inflammation, but also enhance the added value of *Lilium lancifolium* products. Thus, researching methods for rapid and large-scale rooting of *Lilium lancifolium* is of great significance.

[0003] In recent years, most research on the rooting and development of lilies has been based on bulbs and stems. For example, invention patent CN104160805B discloses a method for promoting the germination and rooting of lily bulbs. This method includes the preparation of a simple germination and rooting shed and rooting bed; thawing of lily bulbs; disinfection and treatment with rooting promoters; placement and covering of bulbs with soil; control of temperature and humidity in the germination and rooting shed, maintaining appropriate substrate moisture; and field planting after germination and rooting. Invention patent CN111903519B discloses a method for inducing rooting in large lilies after breaking dormancy. This method involves collecting large lily bulbs that have stopped growing in autumn as explants, sterilizing and treating the bulbs at low temperature, and then placing them under suitable temperature, humidity, and light intensity conditions to induce rooting. Invention patent CN109463282A uses the inner scales of the tuber of *Lilium longiflorum* as explants, inoculated into an induction medium to induce adventitious buds from the *Lilium longiflorum* scales, and then cultured to differentiate into 3-4 cm *Lilium longiflorum* tissue culture seedlings; these seedlings are then inoculated into a rooting medium to induce rooting. However, the above methods mainly focus on lily seedling cultivation, often employing tissue culture or induction culture, which is cumbersome and time-consuming. More importantly, the roots are embedded in the culture medium, making operation inconvenient and failing to meet market demands. Currently, there are no reports on how to quickly and easily obtain a large number of lily roots in a short period of time.

[0004] The above-ground parts of the *Lilium tigrinum* produce numerous specialized small bulbils, commonly known as "bulbils," in the leaf axils. These bulbils, as tiny propagules, are crucial tissue materials for lily seedling cultivation and rapid virus-free propagation. Currently, very few *Lilium* species can produce bulbils naturally, including *Lilium tigrinum*, *Lilium bulbiferum*, *Lilium lancifolium*, and *Lilium tongjiangense*. However, research on bulbil propagation methods often focuses on using bulbils as explants for tissue culture, primarily concerning bulbil germination and cultivation. Furthermore, the vernalization process is lengthy, requiring approximately 5–9 weeks. Research on the direct and rapid rooting of bulbils remains largely unexplored. Therefore, there is an urgent need to find a simple, rapid, and high-volume method for rooting lily bulbils. Summary of the Invention

[0005] To address the aforementioned shortcomings of existing technologies, the present invention aims to provide a method for inducing rapid rooting of lily bulbils and its application, solving the problems of long time cycles for obtaining lily roots and the reliance on tissue culture systems for root induction. The method of the present invention is simple to operate and can obtain a large number of lily roots in a short time, while filling the research gap on direct and rapid rooting of bulbil scales.

[0006] To achieve the above objectives, the present invention adopts the following solution: a method for inducing rapid rooting of lily bulbils, comprising the following steps:

[0007] 1) Use bulbils of lilies (diameter > 1.0cm) as material, perform surface disinfection treatment and air dry naturally for later use;

[0008] 2) Place the disinfected bulbils from step 1) at 4°C for low-temperature pretreatment for 2 weeks;

[0009] 3) Using the bulbils pretreated in step 2) as material, peel off the outer scales and place them in fermented sawdust with a humidity of 15% to 25% for cultivation in the dark. Once the bulbils have rooted, lily roots are obtained. The outer scales of the bulbils are detached tissue.

[0010] Preferably, the disinfection in step 1) is performed by treating with potassium permanganate at a mass fraction of 0.4-0.6% for 5-10 minutes.

[0011] Preferably, the culture temperature in step 3) is 26℃±2℃.

[0012] Another object of the present invention is to provide an application of the above method in the cultivation of lily seedlings.

[0013] Preferably, the lily is lily of the tiger lily, bulbil lily, pale yellow lily, or Tongjiang lily.

[0014] Another object of the present invention is to provide the use of lily root obtained by the above method in the preparation of drugs, foods or cosmetics with anti-melanin synthesis function and / or anti-inflammatory function.

[0015] Preferably, the lily is Lilium tigrinum.

[0016] Compared with the prior art, the present invention has the following beneficial effects:

[0017] 1. This invention provides a method for inducing rapid rooting of lily bulbils. Multifactorial ANOVA was used to analyze the effects of low-temperature pretreatment, culture medium, culture temperature, and their interactions on bulbil scale rooting. Results showed that the interaction between low-temperature pretreatment and the culture medium had the largest impact on the rooting effect of *Lilium tigrinum* bulbil scales; culture temperature also had a significant effect on bulbil scale rooting. The method of this invention is particularly suitable for the rooting of lily bulbil scales. Compared with the control, the rooting rate of *Lilium tigrinum* bulbil scales increased from 60%–70% to 98.89%; root germination was earlier, with an average of 5 roots per bulbil scale and a root length of over 7 cm. Both the number and length of roots showed a significant increase, providing a new method for rapid rooting of lily bulbils.

[0018] 2. The root system of *Lilium tigrinum* obtained using this invention is well-developed, and the entire cultivation cycle is short, requiring only about 10 weeks to rapidly obtain a large number of *Lilium tigrinum* bulbil scales' roots. This fills the research gap in the direct and rapid rooting of lily bulbil scales under in vitro conditions, and can be applied to the preparation of drugs, foods, and cosmetics with anti-melanin synthesis and / or anti-inflammatory functions. It is naturally derived, has no toxic side effects, provides technical support for the development of subsequent products, and enhances the economic value of *Lilium tigrinum*. Simultaneously, this method also provides a new option for the rapid propagation system of *Lilium tigrinum*, which has significant scientific value.

[0019] 3. This invention selects lily bulbils (diameter > 1.0 cm) as material, which is readily available, does not rely on tissue culture or induction medium, and is simple and easy to operate. Rooting is achieved using detached bulbil outer scales, resulting in low cost, high efficiency, high output, and the potential for large-scale production. The pretreatment time is short, requiring only 2 weeks, significantly shortening the cultivation cycle and solving the problem of long vernalization time in lilies, thus demonstrating promising application prospects. Attached Figure Description

[0020] Figure 1 Comparison of bulbil rooting rates under different treatment conditions: a) 2 weeks of culture, b) 4 weeks of culture, c) 6 weeks of culture, d) 8 weeks of culture.

[0021] Figure 2 The growth of the bulbils of *Tilia tigrinosa* is shown below; a to d represent the growth of Comparative Example 1 at 2, 4, 6, and 8 weeks respectively; e to h represent the growth of Example 2 at 2, 4, 6, and 8 weeks respectively; i to l represent the growth of Comparative Example 2 at 2, 4, 6, and 8 weeks respectively; the scale represents 1 cm. Detailed Implementation

[0022] The present invention will be further described in detail below with reference to specific embodiments and accompanying drawings. The bulbils of *Lilium tigrinum* used in the following embodiments were collected in May 2022 from the lily experimental base of the Flower Genetics and Breeding Team at Yangtze Normal University. The fermented sawdust used in the experiment was purchased from Hengjian Ecological Agriculture Cooperative, the peat moss from Luyuan Soil Materials and Fertilizer Co., Ltd., and the humus soil from Chongqing Huaqing Agricultural Technology Co., Ltd.

[0023] Example 1: Optimization of conditions for rapid rooting of lily bulbils

[0024] Healthy, plump, and uniformly sized *Lilium tigrinum* bulbils (diameter > 1.0 cm) were selected and surface-sterilized by soaking in a 0.5% potassium permanganate solution for 5–10 minutes. After rinsing with clean water, they were air-dried at room temperature for later use. The sterilized bulbils were then pretreated. After pretreatment, the outer scales were removed from the bulbils, and they were cultured in a dark medium for 8 weeks. The experiment considered the pretreatment method, culture medium, and culture temperature as factors, taking into account the interactions between different factors (A×B, A×C, B×C, A×B×C). Specific factors and levels are shown in Table 1.

[0025] Table 1

[0026]

[0027] The specific experimental procedure was as follows: First, the bulbils were randomly divided into three groups and subjected to different pretreatments: a room temperature control, a 4℃ treatment for one week, and a 4℃ treatment for two weeks. Then, each group was further divided into three subgroups. The pretreated bulbils from each group were placed in fermented sawdust, peat moss, and a mixed substrate (fermented sawdust: humus: peat moss = 1:1:1) for light-protected cultivation. Each subgroup was further divided into two portions, each containing 30 bulbils, and placed at cultivation temperatures of 20℃ and 26℃ respectively. The humidity of the culture medium in each group was maintained at approximately 20% (they clump together when squeezed but crumble when released). The rooting rate of the bulbils was recorded every two weeks (weeks 2, 4, 6, and 8). The experiment was repeated three times, and the average results were taken. Figure 1 As shown in Table 2.

[0028] The rooting rate of bulbils is calculated using the following formula:

[0029] Rooting rate of bulbils (%) = Number of rooted bulbils / Total number of bulbils * 100;

[0030] Furthermore, the relevant data were organized using Excel 2021, and a multi-factor ANOVA was performed on the data using IBM SPSS Statistics 26 software. Charts were created using Origin 2019b software, and the results are shown in Tables 3 and 4.

[0031] Table 2

[0032]

[0033] Table 3

[0034]

[0035] Table 4

[0036]

[0037] from Figure 1As shown in Table 2, there were significant differences in the rooting rate of *Lysimachia christinae* bulbils under different treatments, with the difference increasing with the extension of culture time. Among the bulbils, after 8 weeks of growth under different culture conditions, treatment 13 showed the best rooting effect with a rooting rate of 98.89%, followed by treatments 3 and 5 with rooting rates of 91.11% and 90.00%, respectively. Treatment 1 had the lowest rooting rate. Analysis of variance (Table 3) showed that among the three single-factor experiments, only culture temperature had a significant effect on the rooting rate of *Lysimachia christinae* bulbils (F = 6.196, p = 0.018), while the other two factors had no significant effect. Among the interactions among the three factors, only the interaction between low-temperature pretreatment and the culture medium had a significant effect on the rooting of *Lysimachia christinae* bulbils (F = 3.314, p = 0.021). The effect size of each culture factor on the rooting rate of *Lysimachia christinae* bulbils was ranked as A×B>A>A×B×C>C>A×C>B×C. Therefore, the order of importance of each factor was A×B>C>A×B×C>A>B>B×C>A×C. Among these factors, 26.9% of the bulbil rooting rate could be explained by the interaction between low-temperature pretreatment and the culture medium, accounting for the largest proportion, followed by culture temperature at 14.7%. The mean values ​​of different levels of low-temperature pretreatment and culture medium combinations (Table 4) showed that the combination of 4℃ treatment for 2 weeks and fermented sawdust had the best effect on the rooting rate of bulbil scales. Based on the above analysis, treating *Lysimachia christinae* bulbils at 4℃ for 2 weeks and then culturing the outer scales in fermented sawdust at 26℃ is the optimal culture method to promote rooting.

[0038] Example 2

[0039] A method for inducing rapid rooting based on lily bulbils includes the following steps:

[0040] 1) Using lily bulbils (diameter > 1.0 cm) as material, soak them in 0.5% potassium permanganate solution for 5-10 minutes for surface disinfection and then air dry at room temperature for later use;

[0041] 2) Place the disinfected bulbils from step 1) at 4°C for low-temperature pretreatment for 2 weeks;

[0042] 3) Peel the outer scales from the bulbils after the pretreatment in step 2) and place them in fermented sawdust with a humidity of 15% to 25% for 8 weeks in the dark at 26°C.

[0043] Comparative Example 1

[0044] 1) Using lily bulbils (diameter > 1.0 cm) as material, soak them in 0.5% potassium permanganate solution for 5-10 minutes for surface disinfection and then air dry at room temperature for later use;

[0045] 2) Peel the outer scales off the bulbils after disinfection in step 1) and place them directly into fermented sawdust with a humidity of 15% to 25% for 8 weeks in the dark at 20°C.

[0046] Comparative Example 2

[0047] 1) Using lily bulbils (diameter > 1.0 cm) as material, soak them in 0.5% potassium permanganate solution for 5-10 minutes for surface disinfection and then air dry at room temperature for later use;

[0048] 2) Place the disinfected bulbils from step 1) at 4°C for a low-temperature pretreatment for 1 week;

[0049] 3) Peel the outer scales off the bulbils after the pretreatment in step 2) and place them directly into peat moss with a humidity of 15% to 25% for 20°C in the dark for 8 weeks.

[0050] The rooting of bulbils in Example 2 and Comparative Examples 1-2 was observed, and the results are as follows: Figure 2 As shown in the figure (the bulbils in the figure are randomly selected at corresponding stages and can reflect the overall rooting status of each experimental group), compared with Comparative Examples 1 and 2, the root germination rate of the bulbils in this embodiment is faster, with an average of 5.05 roots per bulbil (compared to 3.07 and 3.01 roots in Comparative Examples 1 and 2, respectively), and an average root length of 7.41 cm (compared to 5.15 cm and 4.72 cm in Comparative Examples 1 and 2, respectively). It is evident that the method of this invention significantly increases the number and length of roots, and this invention can obtain a large number of *Lilium tigrinum* roots in about 10 weeks.

[0051] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit it. Although the applicant has described the present invention in detail with reference to preferred embodiments, those skilled in the art should understand that any modifications or equivalent substitutions to the technical solutions of the present invention that do not depart from the spirit and scope of the technical solutions of the present invention should be covered within the scope of the claims of the present invention.

Claims

1. A method for inducing rapid rooting of lily bulbils, characterized in that, Includes the following steps: 1) Using lily bulbils as material, surface disinfection is performed with potassium permanganate at a mass fraction of 0.4-0.6% for 5-10 minutes and then air-dried for later use. The diameter of the bulbils is >1.0cm. 2) Place the disinfected bulbils from step 1) at 4°C for low-temperature pretreatment for 2 weeks; 3) Using the bulbils pretreated in step 2) as material, peel off the outer scales and place them in sterile fermented sawdust with a humidity of 15%~25% for 8 weeks at 26℃±2℃ in the dark. Once the bulbils have taken root, the lily root system will be obtained.

2. The application of the method as described in claim 1 in the cultivation of lily seedlings.

3. The application according to claim 2, characterized in that, The lilies mentioned are Lilium tigrinum, Lilium bulbiferum, Lilium lancifolium, or Lilium tongjiangense, which can produce bulbils under natural conditions.

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