Method for regulating abundance of lily stem cells in vitro

Abstract

The present application relates to the field of plant tissue culture technology, and particularly discloses a lily stem cell abundance ex vivo regulation method. The lily bulb is used as the starting material, stem cell magnitude amplification and regulation culture can produce lily tissue organ materials with high stem cell abundance. The present application provides sterile lily materials with high stem cell abundance, which has the characteristics of strong regenerative ability, high proliferation rate, etc., is the basis for protoplast preparation and high-efficiency regeneration, and is suitable for different lily varieties, and provides technical support for cell engineering breeding, seedling scale breeding, etc.

Description

Technical Field

[0001] This invention relates to the field of plant tissue culture technology, specifically disclosing a method for in vitro regulation of lily stem cell abundance. Background Technology

[0002] Lily (Lilium spp.) is a perennial bulbous flowering plant belonging to the genus Lilium in the family Liliaceae. It is known as the "King of Bulbous Flowers" due to its high ornamental value. Its bulbs are rich in starch, protein, vitamins, alkaloids, and other nutrients, making it highly nutritious. Lily foods also possess medicinal properties such as clearing the lungs, moisturizing dryness, nourishing yin, and clearing heat. Given lilies' significant economic value as ornamental, edible, and medicinal plants, there is a high market demand for new lily varieties. Traditional lily breeding methods rely on hybridization, which suffers from problems such as incompatibility in distant hybridization, long breeding cycles, low breeding precision, and difficulty in rapidly and efficiently cultivating target varieties. Cell engineering breeding, achieved through protoplast fusion, has seen few successful reports of lily cell fusion.

[0003] Lilies are herbaceous plants with rapid cell maturation and low stem cell abundance, resulting in extremely low efficiency in protoplast regeneration and cell engineering breeding. While lily protoplast starting materials are widely available, not all materials yield high-yield, highly viable protoplasts. For example, isolating protoplasts from highly differentiated plant organs such as roots is challenging. The degree of differentiation in lily organs affects the totipotency of protoplasts; starting materials with low totipotency produce protoplasts with low totipotency, making it difficult to differentiate into complete plants. Therefore, preparing lily materials with high stem cell abundance and high differentiation potential is of great significance.

[0004] Plant stem cells are a population of cells within plants that possess the potential for self-renewal and multi-directional differentiation. They exist in the apical meristem formed during the embryonic period, the lateral meristem formed after embryonic development, and new growth points established during somatic cell fate transitions in the regeneration process. Plant somatic cells exhibit strong plasticity and, under certain induction conditions, can be reprogrammed and undergo fate transitions to form totipotent and pluripotent stem cells (i.e., plant somatic cells can be reprogrammed to form stem cells or stem cell-like cells), thereby regenerating a complete plant.

[0005] Plant stem cell abundance can be regulated in vitro. The synergistic effect of cytokinins and auxins activates high expression of the WUS gene, which is the molecular basis for the formation of bud stem cells and meristems. Exogenous hormones can effectively increase the quantity of plant stem cells, thereby regulating plant growth and development, accelerating plant propagation, and improving plant regeneration capacity. Using plant tissue culture techniques to obtain lily materials with high stem cell abundance can shorten the lily breeding cycle, increase the propagation coefficient, and compared with callus tissue, lily materials with high stem cell abundance maintain the original superior traits of the variety while improving regeneration capacity.

[0006] High stem cell abundance preparation technology has made great contributions to lily cell engineering breeding and large-scale seedling propagation. Summary of the Invention

[0007] This invention, through research on in vitro culture techniques such as aseptic system establishment, direct adventitious bud generation, adventitious bud subculture proliferation, and stem cell-scale expansion, establishes a highly efficient and rapid propagation system for lilies. It achieves a high enrichment of stem cell content in lily materials and provides a method based on hormone regulation to control the scale of lily stem cells. This method is used to obtain a large number of lily seedlings with strong regenerative capabilities, providing the market with a method for the continuous cultivation of lily seedlings with high stem cell abundance. It also provides technical support for the rapid propagation of lily tissues and the acquisition and regeneration of protoplasts.

[0008] This invention provides a method for in vitro culture of lilies with high stem cell abundance, comprising the following steps:

[0009] (1) Disinfect lily bulbs;

[0010] (2) Induction of adventitious shoots: The sterile explants obtained in step (1) were transferred to MS medium supplemented with 1.0-2.0 mg / L 6-benzylaminopurine (6-BA) + 0.1-0.2 mg / L α-naphthaleneacetic acid (NAA) to induce adventitious shoots in lilies;

[0011] (3) Subculture proliferation and elongation of adventitious shoots: The adventitious shoots obtained in step (2) were inoculated into MS medium containing 0.5-1.5 mg / L 6-BA, 0.1-0.3 mg / L NAA, 0.5-1.5 mg / L 2,4-dichlorophenoxyacetic acid (2,4-D), 0.1-0.3 mg / L kinetin (KT), and 0-0.5 mg / L thiamethoxam (TDZ) to promote the proliferation of lily adventitious shoots;

[0012] (4) Mass expansion of lily stem cells: The material obtained in step (3) was inoculated into MS medium supplemented with 2.0-3.0 mg / L 6-BA and 1.0-2.0 mg / L TDZ to induce mass expansion of lily stem cells.

[0013] Specifically, the disinfection in step (1) is as follows: Select healthy and clean lily scales, wash the lily scales with detergent, rinse them under running water for 2 hours, add 1:1000 potassium permanganate solution, and sonicate at 40kHz for 30 minutes; cut off 1 / 3 of the tip of the scales, and cut the remaining scales longitudinally into strips of 1.5-2 cm for later use. Treat the pretreated material with 75% ethanol solution for 30-60 seconds, 20% sodium hypochlorite solution for 12-18 minutes, and PB disinfectant solution containing 50% plant tissue culture antibacterial agent PPM and 1% benzalkonium bromide for 6-8 minutes. Finally, rinse with sterile water 3-5 times and dry for later use.

[0014] Specifically, the culture time for inducing adventitious bud formation in step (2) is 12-18 days; the culture time for adventitious bud proliferation in step (3) is 45-70 days; the culture time for stem cell-scale expansion in step (4) is 70-80 days, and then the cells are transferred every 45 days.

[0015] Preferably, the culture medium in step (2) is MS medium supplemented with 2.0 mg / L 6-BA and 0.1 mg / L NAA;

[0016] The culture medium in step (3) is MS medium containing 1.0 mg / L 6-BA, 0.2 mg / L NAA, 0.5 mg / L 2,4-D, and 0.2 mg / L KT;

[0017] The culture medium in step (4) is MS medium supplemented with 3.0 mg / L 6-BA and 2.0 mg / L TDZ.

[0018] Specifically, 25-35 g / L of sucrose and 5-6 g / L of agar powder are added to the culture medium to adjust the pH of the culture medium to 5.6-6.0; preferably, 30 g / L of sucrose and 5.5 g / L of agar powder are added to the culture medium to adjust the pH of the culture medium to 5.8.

[0019] More preferably, the cultivation conditions are 25±2℃, light intensity 2000-3000lx, and light exposure 14-18h / d.

[0020] The present invention provides the application of the material obtained by the method described herein in the preparation of lily protoplasts.

[0021] The present invention further provides a method for preparing lily protoplasts, which uses the materials obtained by the method to prepare protoplasts.

[0022] Specifically, the protoplasts are separated by enzymatic hydrolysis. More specifically, the enzymatic hydrolysis method is as follows: the lily material is cut into small pieces, immersed in the enzymatic hydrolysis solution, and incubated in the dark at 20-30℃ for 6-8 hours; after enzymatic hydrolysis, the solution is filtered, the filtrate is centrifuged, the supernatant is removed, and the protoplasts are obtained.

[0023] This invention, through research and exploration, identifies key steps in lily in vitro culture and stem cell scale expansion, and obtains a method to increase the scale of lily stem cells in vitro. In the field of rapid propagation of plant tissues, it can obtain a large amount of lily in vitro material with regenerative capacity. Furthermore, the protoplasts obtained from lily material with high stem cell abundance have the advantages of high yield and high viability, and have a strong advantage in protoplast regeneration. Attached Figure Description

[0024] Figure 1 Lily bulb.

[0025] Figure 2 Lily adventitious buds.

[0026] Figure 3 Lily adventitious buds proliferate.

[0027] Figure 4 Massive expansion of lily stem cells.

[0028] Figure 5 Paraffin sections of high stem cell lily material

[0029] Figure 6 Paraffin sections of lily bulbs (control material)

[0030] Figure 7 Lily bulb (control material) protoplast vitality

[0031] Figure 8 High stem cell lily material protoplast viability. Detailed Implementation

[0032] The present invention will be further illustrated below through a detailed description of specific embodiments, but this is not intended to limit the invention and is merely an illustrative example.

[0033] The lily basal culture medium was MS (Murashige and Skoog 1962) medium supplemented with different ranges of plant hormones, pH 5.8, and sterilized at 121℃ for 15-25 minutes. This medium was used for adventitious shoot induction, proliferation, and stem cell-scale expansion. Culture conditions: All media were supplemented with 30 g / L sucrose, 0.1 g / L inositol, and 5.5 g / L agar powder, and the pH was adjusted to 5.8. Culture conditions were 25 ± 2℃, under light (50 μmol·m⁻¹). -2 ·s -1 16h / d.

[0034] Example 1

[0035] (1) Preparation of explant materials

[0036] Healthy lily scales were selected from lily bulbs as initial explants. Figure 1 Select healthy, clean lily scales, wash the removed scales with detergent, rinse under running water for 2 hours, add 1:1000 potassium permanganate solution, and sonicate at 40kHz for 30 minutes. Cut off the tip of the scales by 1 / 3, and cut the remaining scales longitudinally into 1.5-2 cm strips for later use. Place the pretreated material into a sterile Erlenmeyer flask, transfer it to a laminar flow hood, and treat with 75% ethanol solution for 30-60 seconds, 20% sodium hypochlorite solution for 12-18 minutes, and PB disinfectant solution containing 50% plant tissue culture antibacterial agent PPM and 1% benzalkonium bromide for 2-8 minutes. Finally, rinse with sterile water 3-5 times and blot dry for later use.

[0037] The experimental results (Table 1) show that: if the treatment time of PB disinfectant is too short, the contamination rate is high; the effect of PB disinfectant treatment time of 6-8 min is better. Under the same PB disinfectant treatment time, the contamination rate of 20% sodium hypochlorite treatment time is low, but the inactivation rate is high. Among them, the combination of 75% ethanol 30s + 20% sodium hypochlorite 15min + 6min PB has the best disinfection effect and the highest survival rate.

[0038] Table 1. Effects of different disinfection methods on the viability of lily bulbs

[0039] Disinfection plan Pollution rate % Survival rate % 75% ethanol 30s + 20% sodium hypochlorite 12min + 8min PB 13.3 83.3 75% ethanol 30s + 20% sodium hypochlorite 15min + 6min PB 10.0 86.6 75% ethanol 60s + 20% sodium hypochlorite 18min + 6min PB 6.7 83.3 75% ethanol 60s + 20% sodium hypochlorite 20min + 4min PB 23.3 66.6 75% ethanol 60s + 20% sodium hypochlorite 20min + 2min PB 56.6 23.3

[0040] (2) Adventitious bud induction

[0041] The obtained sterile explants were inoculated into MS medium supplemented with 0.0-2.0 mg / L 6-BA and 0.0-0.2 mg / L NAA for adventitious bud induction. After 15-20 days of culture, adventitious bud germination was observed (Table 2). Adventitious buds could be induced in lily bulbs in MS medium supplemented with 0.0-2.0 mg / L 6-BA and 0.0-0.2 mg / L NAA. Adding different combinations of plant hormones to the MS medium was beneficial for lily adventitious bud induction, and adventitious buds were induced after approximately 14 days. Figure 2 Considering the adventitious bud induction rate and average number of buds of the three lily varieties, the culture medium with 2 mg / L 6-BA and 0.1 mg / L NAA showed the best induction effect, with an average adventitious bud induction rate of 96.16% and an average number of buds of 4 per explant.

[0042] Table 2. Effects of different hormone formulations on adventitious shoot induction in explants

[0043]

[0044]

[0045] (3) Lily adventitious bud propagation culture

[0046] The clustered shoots obtained from the initial culture were isolated and transferred to an adventitious shoot proliferation medium for adventitious shoot proliferation culture. The proliferation of adventitious shoots was statistically analyzed after 60 days. Figure 3 Considering all factors, the combination of 1.0 mg / L 6-BA, 0.2 mg / L NAA, 0.5 mg / L 2,4-D, and 0.2 mg / L KT resulted in the best adventitious bud proliferation effect among the three lily varieties.

[0047] Table 3. Adventitious bud proliferation of lilies

[0048]

[0049] (4) Mass expansion of lily stem cells

[0050] The lily propagation seedlings obtained in step (3) were transferred to stem cell-scale expansion medium. MS medium was used as the basal medium, with 1.0-4.0 mg / L 6-BA and 0.0-3.0 mg / L TDZ added. The culture was carried out for 70-80 days, followed by transfer every 45 days. Histological observation of the lily materials revealed that the cells in lily bulbs that had not undergone stem cell-scale expansion (after 80 days of culture on MS0 medium) were larger, irregularly arranged, and had smaller nuclei that were less easily stained. Figure 6 The stem cell abundance expansion material has small cell volume, regular and compact arrangement, clear boundaries, dense cytoplasm, large and easily stained nuclei, and vigorous cell division ability. Figure 5 Considering the histological and morphological conditions of the three lily varieties, the culture medium supplemented with 3.0 mg / L 6-BA + 2.0 mg / L LTDZ showed the best effect on the expansion of lily stem cells.

[0051] Table 4. Effects of different plant hormone combinations on the histological and morphological aspects of lily stem cell expansion.

[0052]

[0053] Example 2: The effect of lily stem cell quantity on protoplast quality

[0054] In Example 1, the lily bulbs used in steps (3) and (4) were used to separate protoplasts using an enzymatic hydrolysis method. Specifically, the lily tissue was placed in a culture dish, chopped with a sharp blade until no large pieces of tissue remained, and immersed in an enzymatic hydrolysis solution (the hydrolysis solution was a CPW solution containing 0.2-0.5 mol / L mannitol, 0.5-4.0 m / v% cellulase, 0.1-0.5 m / v% pectinase, and 0.5 m / v% dissociation enzyme; 1 mL of hydrolysis solution was added for every 100 mg of lily tissue) for enzymatic hydrolysis. The solution was incubated in the dark at 25°C for 6-8 hours. After hydrolysis, the solution was filtered, the filtrate was centrifuged, and the supernatant was discarded to obtain protoplasts. Protoplast viability was then detected using fluorescein diacetate (FDA) staining. Protoplast viability (%) = number of green fluorescent protoplasts / total number of protoplasts × 100%. It was found that the protoplast viability obtained from highly stem cell-derived lily bulbs was above 97%. Figure 8 The viability of protoplasts isolated from lily materials that have not been expanded to stem cell scale is only around 46%. Figure 7 The increased abundance of lily stem cells significantly improved the viability of lily protoplasts.

Claims

1. A method for in vitro culture of lilies with high stem cell abundance, comprising the following steps, (1) Disinfecting lily bulbs; (2) Induction of adventitious shoots: The sterile explants obtained in step (1) were transferred to MS medium supplemented with 1.0-2.0 mg / L 6-benzylaminopurine (6-BA) + 0.1-0.2 mg / L α-naphthaleneacetic acid (NAA) to induce adventitious shoots of lily; (3) Subculture proliferation and elongation of adventitious shoots: The adventitious shoots obtained in step (2) were inoculated into MS medium containing 0.5-1.5 mg / L 6-BA, 0.1-0.3 mg / L NAA, 0.5-1.5 mg / L 2,4-dichlorophenoxyacetic acid 2,4-D, 0.1-0.3 mg / L kinetin KT, and 0-0.5 mg / L thiamethoxam TDZ to promote the proliferation of lily adventitious shoots; (4) Mass expansion of lily stem cells: The material obtained in step (3) was inoculated into MS medium supplemented with 2.0-3.0 mg / L 6-BA and 1.0-2.0 mg / L TDZ to induce mass expansion of lily stem cells.

2. The method as described in claim 1, characterized in that, The culture conditions were 25±2℃, light intensity 2000-3000lx, and light exposure 16 h / d.

3. The method as described in claim 1, characterized in that, The disinfection process in step (1) is as follows: Select healthy, clean lily scales, wash the removed scales with detergent, rinse under running water for 2 hours, add 1:1000 potassium permanganate solution, and sonicate at 40kHz for 30 minutes. Cut off the tip 1 / 3 of the scales, and longitudinally cut the remaining scales into 1.5-2 cm strips for later use. Treat the pretreated material with 75% ethanol solution for 30-60 seconds, 20% sodium hypochlorite solution for 12-18 minutes, and PB disinfectant solution containing 50% plant tissue culture antibacterial agent PPM and 1% benzalkonium bromide for 6-8 minutes. Finally, rinse with sterile water 3-5 times and pat dry for later use.

4. The method as described in claim 1, characterized in that, The culture time for inducing adventitious bud formation in step (2) is 12-18 days; the culture time for adventitious bud proliferation in step (3) is 45-70 days; the culture time for stem cell-scale expansion in step (4) is 70-80 days, and then the cells are transferred every 45 days.

5. The method as described in claim 1, characterized in that, The culture medium in step (2) is MS medium supplemented with 2 mg / L 6-BA and 0.1 mg / L NAA; The culture medium in step (3) is MS medium containing 1 mg / L 6-BA, 0.2 mg / L NAA, 0.5 mg / L 2,4-D, and 0.2 mg / L KT; The culture medium in step (4) is MS medium supplemented with 3 mg / L 6-BA and 2 mg / L TDZ.

6. The method according to any one of claims 1 to 5, characterized in that, Add 25-35 g / L sucrose and 5-6 g / L agar powder to the culture medium and adjust the pH of the culture medium to 5.6-6.0; preferably, add 30 g / L sucrose and 5.5 g / L agar powder to the culture medium and adjust the pH of the culture medium to 5.

8.

7. The method according to any one of claims 1 to 5, characterized in that, The cultivation conditions were 25±2℃, light intensity 2000-3000lx, and light exposure 14-18 h / d.

8. The use of the material obtained by the method according to any one of claims 1 to 7 in the preparation of lily protoplasts.

9. A method for preparing lily protoplasts, characterized in that, Protoplasts are prepared using the material obtained by the method as described in any one of claims 1 to 7.

10. The method as described in claim 9, characterized in that, Specifically, the protoplasts are separated by enzymatic hydrolysis. More specifically, the enzymatic hydrolysis method is as follows: the lily material is cut into small pieces, immersed in the enzymatic hydrolysis solution, and incubated in the dark at 20-30℃ for 6-8 h; after enzymatic hydrolysis, the solution is filtered, the filtrate is centrifuged, the supernatant is removed, and the protoplasts are obtained.

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