A method for promoting differentiation of lycoris squamiferum scales and reducing scale browning

Abstract

The present application relates to the technical field of plant tissue culture, and particularly relates to a method for promoting scale differentiation of Lilium auratum and reducing scale browning. ‑1 When NAA is 0.05 mg / L ‑1 , TDZ is 0.25 mg / L ‑1 , and AC is 6 g / L, the scale differentiation rate of Lilium auratum can reach 71.66%±6.00ab, the browning rate is reduced to 26.66%±6.00c, and the bulb growth is significantly promoted, and the bulb size can reach (0.52 cm±0.01ab)×(0.4 cm±0.02a), which can provide support for efficient propagation of Lilium auratum bulbs.

Description

Technical Field

[0001] This invention relates to the field of plant tissue culture technology, specifically to a method for promoting scale differentiation and reducing scale browning in *Lilium davidii*. Background Technology

[0002] Dragon Tooth Lily (Lilium brownii var. viridulum) is a perennial herbaceous plant belonging to the genus Lilium in the family Liliaceae. It is a variant of wild lily (Lilium brownii) and is named for its large, tooth-like scales. It is mainly produced in Shaoyang and Longhui in Hunan Province, and Wanzai and Taihe in Jiangxi Province. It was among the first batch of food and medicine homologous products announced by the Ministry of Health and is highly favored by consumers in Southeast Asian countries and regions. In production, Dragon Tooth Lily is mainly propagated asexually through scale cuttings and bulb division. However, this method leads to genetic degeneration, significantly reducing yield and quality. Furthermore, the breeding process of Dragon Tooth Lily suffers from a low differentiation coefficient and a long propagation cycle. Therefore, high-quality and efficient bulb propagation is the only way to solve the development problem of the Dragon Tooth Lily industry.

[0003] Plant tissue culture is an important research technique and method in life sciences. It can accelerate the propagation of lilies, shorten the growth cycle of lily bulbs, and compensate for the shortcomings of bulb propagation, thus being widely used in the commercialization and industrialization of lilies. In recent years, there have been numerous research reports on lily tissue culture both domestically and internationally. Lily tissue culture often uses scales and stem segments as explants, adding hormones such as 6-BA, NAA, and 2,4-D, either alone or in combination, to promote the induction, proliferation, and enlargement of lily bulbs. For example, Chinese invention patent CN 107396835 A discloses a method for rapid propagation of edible lilies. This invention significantly improves the adventitious bud induction effect of explants from superior lily varieties by determining the culture medium and conditions for each stage of explant proliferation, resulting in a high adventitious bud differentiation rate and a large number of differentiations. However, this method only promotes lily scale differentiation; the browning of scales still exists during lily propagation. Furthermore, the lily genome is large, highly heterozygous, and exhibits varying ploidy among varieties and significant differences between different hybrid lines. Therefore, the required hormone treatment combinations for different lily varieties are not the same, which limits the widespread application of this method. Summary of the Invention

[0004] In view of the above-mentioned shortcomings in the prior art, the purpose of the present invention is to provide a method for promoting scale differentiation and reducing scale browning in Lilium draco.

[0005] To achieve the above-mentioned objectives, the technical solution adopted by this invention is as follows:

[0006] A method for promoting scale differentiation and reducing scale browning in *Lilium draco* is provided, which is achieved by setting up a culture medium and culture conditions; the culture medium and culture conditions are: MS + NAA 0.05 mg·L⁻¹. -1 +TDZ 0.25mg·L -1 +KT 0.05mg·L -1 +AC 6g·L -1 pH 6.0.

[0007] Furthermore, the specific steps include:

[0008] Step 1: Select the clean, spotless scales from the center of the dragon tooth lily bulb and wash them thoroughly under running water;

[0009] Step 2: Prepare culture medium: MS + NAA 0.05 mg·L -1 +TDZ 0.25mg·L -1 +KT 0.05mg·L -1 +AC6g·L -1 pH 6.0, culture medium should be sterilized at high temperature before use;

[0010] Step 3: Place the scales in a sterile empty bottle and sterilize them under ultraviolet light for 15 minutes in a clean bench. Then treat them with 75% alcohol for 30 seconds, followed by treatment with 10% NaClO for 15 minutes. Rinse them with sterile water 3 times, 3 minutes each time.

[0011] Step 4: Remove the transparent part around the scales, cut the scales into two parts, and inoculate them into the culture medium with the concave side facing up;

[0012] Step 5: Observe the differentiation and browning of lily scales after 20 days, calculate the differentiation rate, and determine the size of the seed bulb.

[0013] Furthermore, activated carbon (AC) needs to be added to the culture medium at the end.

[0014] The beneficial effects of this invention are as follows:

[0015] This invention proposes a method to promote scale differentiation and reduce scale browning in *Lilium davidii*, which can effectively increase the scale differentiation rate and reduce scale browning, shorten the seed bulb growth time, and significantly promote seed bulb growth. The method involves MS + NAA 0.05 mg·L⁻¹. -1 +TDZ 0.25mg·L -1 +KT 0.05mg·L -1 The optimal culture medium for inducing scale differentiation in *Lilium draco* seed bulbs was found to be 81.67% ± 3.33 a. Adding 6 g·L⁻¹ of this medium resulted in a scale differentiation rate of 81.67% ± 3.33 a. -1The differentiation rate after AC was 71.66% ± 6.00ab, which was not significantly different from the control. However, the browning rate was significantly reduced to 26.66% ± 6.00c, and the bulb size reached (0.52cm ± 0.01ab) × (0.4cm ± 0.02a) (length × width), which was significantly higher than other groups. This can provide support for the efficient propagation of Lilium longiflorum bulbs. Attached Figure Description

[0016] Figure 1 Table 1 shows the scale growth of *Lilium draco* under different differentiation culture medium conditions in the examples, after 20 days; the numbers in the figure represent the treatment numbers.

[0017] Figure 2 Table 2 shows the growth of *Lilium lancifolium* under different anti-browning culture media in the examples; the numbers in the figure represent different concentrations of activated carbon. Detailed Implementation

[0018] The specific embodiments of the present invention are described below to enable those skilled in the art to understand the present invention. However, it should be understood that the present invention is not limited to the scope of the specific embodiments. For those skilled in the art, various changes are obvious as long as they are within the spirit and scope of the present invention as defined and determined by the appended claims. All inventions utilizing the concept of the present invention are protected.

[0019] Test case

[0020] This experiment first performed a scale differentiation test on *Lilium draco* to screen out the optimal differentiation medium and culture conditions: MS + NAA 0.05 mg·L⁻¹. -1 +TDZ 0.25mg·L -1 +KT 0.05mg·L -1 The pH was set to 6.0, and then a browning test was conducted. The browning test involved adding different concentrations of activated carbon to the optimal culture medium for the differentiation test.

[0021] The specific steps of the differentiation experiment are as follows:

[0022] Step 1: Select the clean, spotless scales from the center of the dragon tooth lily bulb and wash them thoroughly under running water;

[0023] Step 2: Prepare scale differentiation culture media for different hormone treatments, and use the culture media after high-temperature sterilization;

[0024] Step 3: Place the scales in a sterile empty bottle and sterilize them under ultraviolet light for 15 minutes in a clean bench. Then treat them with 75% alcohol for 30 seconds, followed by treatment with 10% NaClO for 15 minutes. Rinse them with sterile water 3 times, 3 minutes each time.

[0025] Step 4: Remove the transparent part around the scales, cut the scales into two parts, with the concave side facing up, and inoculate them into different culture media;

[0026] Step 5: Observe the differentiation of lily scales after 20 days and calculate the differentiation rate.

[0027] Table 1

[0028]

[0029] The specific steps of the browning test are as follows:

[0030] Step 1: Select the clean, spotless scales from the center of the dragon tooth lily bulb and wash them thoroughly under running water;

[0031] Step 2: In differentiation medium MS + NAA 0.05 mg·L -1 +TDZ 0.25mg·L -1 +KT 0.05mg·L -1 Different concentrations of activated carbon AC were added (Table 2). AC was to be added to the culture medium last, and the culture medium was to be used after high-temperature sterilization.

[0032] Step 3: Place the scales in a sterile empty bottle and sterilize them under ultraviolet light for 15 minutes in a clean bench. Then treat them with 75% alcohol for 30 seconds, followed by treatment with 10% NaClO for 15 minutes. Rinse them with sterile water 3 times, 3 minutes each time.

[0033] Step 4: Remove the transparent part around the scales, cut the scales into two parts, and inoculate them into the culture medium with the concave side facing up;

[0034] Step 5: Observe the differentiation and browning of lily scales after 20 days, calculate the differentiation rate, and determine the size of the seed bulb.

[0035] Table 2

[0036]

[0037] In summary, this invention, through observation of scale differentiation and browning under different culture medium conditions, found that when NAA is 0.05 mg·L⁻¹, the optimal concentration of NAA is significantly higher than that of scales. -1 TDZ is 0.25 mg·L -1 KT is 0.05 mg·L -1 When AC is 6 g / L, the scale differentiation rate of *Lilium longiflorum* can reach 71.66% ± 6.00ab, the browning rate is reduced to 26.66% ± 6.00c, and the growth of seed bulbs is significantly promoted. The seed bulb size can reach (0.52cm ± 0.01ab) × (0.4cm ± 0.02a) (length × width), which can provide support for the efficient propagation of *Lilium longiflorum* seed bulbs.

[0038] It will be apparent to those skilled in the art that the present invention is not limited to the details of the exemplary embodiments described above, and that the invention can be implemented in other specific forms without departing from the spirit or essential characteristics of the invention. Therefore, the embodiments should be considered in all respects as exemplary and non-limiting, and the scope of the invention is defined by the appended claims rather than the foregoing description. Thus, it is intended that all variations falling within the meaning and scope of equivalents of the claims be included within the present invention.

[0039] Furthermore, it should be understood that although this specification describes embodiments, not every embodiment contains only one independent technical solution. This narrative style is merely for clarity. Those skilled in the art should consider the specification as a whole, and the technical solutions in each embodiment can also be appropriately combined to form other embodiments that can be understood by those skilled in the art.

Claims

1. A method for promoting scale differentiation and reducing scale browning in *Lilium draco*, characterized in that, White, spotless scales from the center of *Lilium davidii* bulbs were selected as explants, and culture conditions were established. The culture medium and conditions were: MS + NAA 0.05 mg·L⁻¹. -1 +TDZ 0.25mg·L -1 +KT 0.05 mg·L -1 +AC 6g·L -1 pH 6.

0.

2. The method for promoting scale differentiation and reducing scale browning in *Lilium draco* according to claim 1, characterized in that, Specifically, the following steps are included: Step 1: Select the clean, spotless scales from the center of the dragon tooth lily bulb and wash them thoroughly under running water; Step 2: Prepare culture medium: MS + NAA 0.05 mg·L -1 +TDZ 0.25mg·L -1 +KT 0.05 mg·L -1 +AC 6g·L -1 pH 6.0, culture medium should be sterilized at high temperature before use; Step 3: Place the scales in a sterile empty bottle and sterilize them under ultraviolet light for 15 min in a clean bench. Then treat them with 75% alcohol for 30 s, then with 10% NaClO for 15 min, and rinse them with sterile water 3 times for 3 min each time. Step 4: Remove the transparent parts around the scales, cut the scales into two parts, and inoculate them into the culture medium with the concave side facing up.

3. The method for promoting scale differentiation and reducing scale browning in *Lilium draco* according to claim 1 or 2, characterized in that, Activated carbon (AC) needs to be added to the culture medium last.

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