A double-layer rooting and seedling strengthening medium for quince tissue culture seedlings and a method for rooting and seedling raising of quince tissue culture seedlings
By using a double-layer rooting and strengthening culture medium for quince tissue culture seedlings and a specific light hardening method, the problems of long rooting time and low survival rate in quince tissue culture were solved, enabling rapid propagation and efficient transplanting.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- Filing Date
- 2025-10-14
- Publication Date
- 2026-05-15
AI Technical Summary
Existing quince tissue culture methods involve long rooting times, leaf tip dieback, and timely transplanting after rooting with low survival rates. Traditional propagation methods are not suitable for large-scale application.
A double-layer rooting and seedling strengthening medium was used for quince tissue culture seedlings. The upper layer consisted of 1/2 MS medium with 6.8~7.2 g/L agar, 28~32 g/L sucrose, and 0.8~1.2 mg/L NAA, while the lower layer consisted of MS medium with 6.8~7.2 g/L agar, 28~32 g/L sucrose, and 0.4~0.6 mg/L 6-BA. Combined with specific light and hardening methods, root growth and strong seedlings were promoted.
By shortening the rooting cycle and increasing the rooting rate and survival rate, rapid propagation and efficient transplanting of quince tissue culture seedlings have been achieved.
Smart Images
Figure CN121128599B_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the field of plant tissue culture technology, and in particular to a double-layer rooting and strengthening culture medium for quince tissue culture seedlings and a method for rooting and hardening quince tissue culture seedlings. Background Technology
[0002] Quince (Cydonia oblonga), belonging to the genus Cydonia in the family Rosaceae, is also known as wood pear, or "biye" in Uyghur. Its fruit has a rich aroma and is rich in vitamins, minerals, and dietary fiber. It can be used to make jams, juices, and traditional Chinese medicine, and has high economic value and market prospects.
[0003] Traditional propagation methods for quince are diverse, including division, seed propagation, cuttings, layering, grafting, and bud grafting. Previously, fruit growers mostly used division, but due to limited material availability, it was unsuitable for large-scale propagation. Plant tissue culture technology provides an effective way for rapid crop propagation and germplasm resource preservation. This technology can achieve rapid, virus-free, and high-throughput propagation of superior varieties, theoretically overcoming many shortcomings of traditional propagation methods. However, in the tissue culture process of quince, seedling rooting and hardening-off transplanting are two crucial technical bottlenecks. Existing conventional tissue culture rooting cultures typically use a single-layer culture medium, which has drawbacks such as long rooting times and leaf tip dieback. After rooting, timely transplanting is necessary; otherwise, the seedlings become increasingly weak and have a low survival rate after transplanting. Therefore, to address the problem of low quince propagation rates, it is necessary to provide a rapid quince tissue propagation technology with a short rooting cycle and high rooting and survival rates. Summary of the Invention
[0004] The purpose of this invention is to provide a double-layer rooting and strengthening culture medium for quince tissue culture seedlings and a method for rooting and hardening quince tissue culture seedlings.
[0005] To achieve the above-mentioned objectives, the present invention provides the following technical solution:
[0006] This invention provides a double-layer rooting and seedling strengthening culture medium for quince tissue culture seedlings. The upper layer of the double-layer rooting and seedling strengthening culture medium consists of: 1 / 2 MS + 6.8~7.2 g / L agar + 28~32 g / L sucrose + 0.8~1.2 mg / L NAA; the upper culture medium can induce rooting.
[0007] The lower layer of the double-layer rooting and seedling strengthening medium consists of: MS + 6.8~7.2 g / L agar + 28~32 g / L sucrose + 0.4~0.6 mg / L 6-BA. The lower layer of the medium is supplemented with cytokinin (6-BA) to promote plant growth. When the roots penetrate into this layer, 6-BA can effectively activate the apical meristem, promote stem elongation and leaf primordia differentiation.
[0008] This invention also provides a method for rooting and hardening off quince tissue culture seedlings, comprising the following steps:
[0009] S1. After 4 weeks of subculture, select robust tissue culture seedlings, remove the swollen callus tissue and leaves from the lower part of the tissue culture seedlings, and inoculate them onto the double-layer rooting and strengthening culture medium for quince tissue culture seedlings.
[0010] After inoculation, the cells were first cultured in the dark at 24.5~25.5℃ for 5 days, and then cultured for another 2~3 days under a light intensity of 180~220 lx and a photoperiod of 14 L / 10D. Finally, the cells were transferred to a light intensity of 1800~2200 lx and a photoperiod of 14 L / 10D.
[0011] S2. Starting from inoculation, after 24-26 days of cultivation, open the bottle mouth of the rooted seedlings by 1 / 2 for hardening for 2-3 days, then open it completely for another 2-3 days. The hardening temperature is 23-27℃, the light intensity is 1800-2200 1x, and the photoperiod is 14L / 10D. During the hardening period, add a small amount of sterile water to the bottle. The main purpose is to create a transitional environment with gradually decreasing humidity, thereby promoting the recovery of the cuticle and stomatal function of the seedling leaves.
[0012] S3. Remove the tissue culture seedlings from the culture medium, wash off the culture medium from the roots, then soak the roots of the tissue culture seedlings in a carbendazim solution for 28-30 seconds before transplanting them into the cultivation substrate.
[0013] Preferably, the robust tissue culture seedlings are those with a height ≥3 cm, a stem diameter ≥0.3 mm, and no wilting leaves.
[0014] Preferably, the cultivation substrate comprises vermiculite, nutrient soil, and perlite in a volume ratio of 1:1:1.
[0015] Preferably, before transplanting the tissue culture seedlings, the cultivation substrate is sterilized with a carbendazim solution, wherein the volume ratio of the carbendazim solution to the cultivation substrate is 1:9~11.
[0016] Preferably, the carbendazim solution is a 900-1100 times diluted solution of carbendazim.
[0017] Preferably, after transplanting, the seedlings are kept in a closed incubator for 9-11 days, with the substrate moisture controlled at 83%-87%, the temperature controlled at 24.5-25.5℃, the air humidity at 48-52%, and the photoperiod at 16L / 8D. After 10 days, the incubator is removed, and the seedlings are transplanted to the field when they reach a height of 15-20cm.
[0018] By adopting the above technical solution, the present invention has the following beneficial effects: The present invention constructs a composite culture medium system with upper and lower layers, which connects the two key stages of rooting and seedling strengthening in time and separates them in space, realizing precise temporal control of the two key developmental stages of tissue culture seedlings, namely "rooting first and then strengthening seedlings". This solves the problem of "seedlings dying easily after rooting and roots becoming coarse and corky" in traditional single-layer culture medium, which can improve the rooting rate, transplant survival rate and significantly improve production efficiency. Attached Figure Description
[0019] Figure 1 This is a diagram of tissue culture seedlings after rooting culture in Example 1.
[0020] Figure 2 This is a root system diagram of the tissue culture seedlings after rooting culture in Example 1.
[0021] Figure 3 This is a picture of tissue culture seedlings transplanted 15 days ago in Example 1.
[0022] Figure 4 This is a picture of tissue culture seedlings transplanted 45 days ago in Example 1. Detailed Implementation
[0023] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.
[0024] Example 1
[0025] 1. Preparation of a double-layer rooting and seedling strengthening medium for quince tissue culture seedlings:
[0026] The upper (rooting layer) culture medium consisted of 1 / 2 MS medium + 7.0 g / L agar + 30 g / L sucrose + 1.0 mg / L NAA, pH=6.3; the thickness of the upper culture medium was 1.5 cm.
[0027] The lower layer (seedling layer) culture medium consisted of MS + 7.0 g / L agar + 30 g / L sucrose + 0.5 mg / L 6-BA, pH=6.3; the upper layer culture medium was 1.5 cm thick.
[0028] First, pour the lower culture medium into the culture bottle. After the bottom layer solidifies, pour the upper culture medium at 50°C into the clean bench, avoiding scalding the bottom layer. After it solidifies, you have the desired product.
[0029] 2. Double-layer rooting and seedling cultivation
[0030] Four weeks after subculturing, select robust tissue culture seedlings (height ≥3 cm or more, stem diameter ≥0.3 mm, uniform leaf size, dark green leaf color, and no wilting). Remove the swollen callus tissue at the lower part of the plant, trim excess leaves at the base, and inoculate them into culture bottles (6 cm in diameter, 11 cm in height) containing the above-mentioned culture medium for rooting culture. After inoculation, culture in the dark at 24.5~25.5℃ for 5 days, then move them out of the dark environment and continue to culture for 2 days under conditions avoiding direct light (light intensity approximately 200 lx, photoperiod 14 L / 10 D) as a light adaptation buffer. Subsequently, transfer to the light culture stage with a light intensity of 2000 lx and a photoperiod of 14 L / 10 D.
[0031] 3. Open the lid to harden the seedlings;
[0032] After 25 days, the seedlings underwent hardening-off. At this point, the rooting rate of the rooted tissue culture seedlings reached 93.3%, with each seedling having 12.5 roots and an average root length of approximately 2.7 cm. Hardening-off was performed by first opening the bottle halfway open for 2 days, then opening it completely for another 2 days. During hardening-off, a small amount of sterile water was added to the bottle to create a gradually decreasing humidity transitional environment, thereby promoting the recovery of the seedling leaf cuticle and stomatal function.
[0033] 4. Transplanting;
[0034] The cultivation substrate was mixed in a ratio of vermiculite: nutrient soil: perlite = 1:1:1 (v / v). Then, 10% of the dry substrate volume was treated with a 1000-fold diluted carbendazim solution for sterilization. After thorough mixing, water was added and stirred until the substrate reached a consistency where it could be formed into a clump when squeezed but crumble into several pieces when released. The tissue culture seedlings were removed from the culture bottles, and any residual culture medium was washed off the roots. The roots were then soaked in a 1000-fold diluted carbendazim solution for 30 seconds before transplanting the seedlings into seedling trays containing the substrate. The roots were ensured to be spread out within the substrate, while avoiding contact between the bottom leaves and the substrate. A tray was placed at the bottom of the seedling trays, and water was added through the tray to maintain substrate moisture. Initially, the substrate humidity was controlled at approximately 85%. After transplanting, the seedlings were placed in a seedling incubator for 10 days, with the temperature controlled at 25±0.5℃, humidity at 50%, and a photoperiod of 16L / 8D. Fifteen days after transplanting, begin applying a water-soluble fertilizer containing macro-elements (trade name: Huaduoduo No. 1; manufacturer: Shandong Meigao International Chemical Co., Ltd.). The fertilizer-to-water volume ratio is 1:5000. Add 0.2 mg / L NAA (naphthaleneacetic acid) to the water-soluble fertilizer to promote root growth, and irrigate every 5 days. Once the seedlings reach a height of 10 cm, apply the water-soluble fertilizer + 0.2 mg / L NAA every 10 days. When the seedlings reach a height of 15-20 cm, transplant them to the field.
[0035] Comparative Example 1
[0036] Unlike Example 1, a monolayer culture medium was used for rooting and seedling cultivation. The composition of the monolayer culture medium was: MS + 7.0 g / L agar + 30 g / L sucrose + 0.3 mg / L IBA.
[0037] Comparative Example 2
[0038] Unlike Example 1, a monolayer culture medium was used for rooting and seedling cultivation. The composition of the monolayer culture medium was: 1 / 2 MS + 7.0 g / L agar + 30 g / L sucrose + 0.6 mg / L IBA.
[0039] Comparative Example 3
[0040] Unlike Example 1, a monolayer culture medium was used for rooting and seedling cultivation. The composition of the monolayer culture medium was: 1 / 2 MS + 7.0 g / L agar + 30 g / L sucrose + 1.0 mg / L NAA.
[0041] Comparative Example 4
[0042] Unlike Example 1, a monolayer culture medium was used for rooting and seedling cultivation. The composition of the monolayer culture medium was: 1 / 2 MS + 7.0 g / L agar + 30 g / L sucrose + 1.3 mg / L NAA.
[0043] Comparative Example 5
[0044] Unlike Example 1, a double-layer culture medium was used for rooting and seedling cultivation.
[0045] The upper culture medium was: 1 / 2 MS + 7.0 g / L agar + 30 g / L sucrose + 0.3 mg / L IBA.
[0046] The lower culture medium was: MS + 7.0 g / L agar + 30 g / L sucrose + 0.2 mg / L 6-BA.
[0047] Comparative Example 6
[0048] Unlike Example 1, a double-layer culture medium was used for rooting and seedling cultivation.
[0049] The upper culture medium was: 1 / 2 MS + 7.0 g / L agar + 30 g / L sucrose + 0.6 mg / L IBA.
[0050] The lower culture medium was: MS + 7.0 g / L agar + 30 g / L sucrose + 0.4 mg / L 6-BA.
[0051] Comparative Example 7
[0052] Unlike Example 1, a double-layer culture medium was used for rooting and seedling cultivation.
[0053] The upper culture medium was: 1 / 2 MS + 7.0 g / L agar + 30 g / L sucrose + 1.3 mg / L NAA.
[0054] The lower culture medium was: MS + 7.0 g / L agar + 30 g / L sucrose + 0.7 mg / L 6-BA.
[0055] Experimental Example
[0056] The experiment was conducted on April 1, 2025. Quince tissue culture seedlings subcultured for 4 weeks were subjected to rooting, hardening-off, and transplanting according to the methods described in Example 1 and Comparative Examples 1-7. All experimental materials were quince tissue culture seedlings from the same batch. Each group had 7 culture bottles, with 5 quince tissue culture seedlings in 5 bottles and 4 quince tissue culture seedlings in 2 bottles, for a total of 30 seedlings. After 30 days of culture, at transplanting, the number of rooted seedlings, rooting rate, average number of roots, and root length were recorded, and the plant condition was observed. The results are shown in Table 1.
[0057] Table 1. Rooting status of quince tissue culture seedlings in each group.
[0058] Group Number of inoculated plants Number of rooted plants Rooting rate (%) Average number of roots (stalks) Root length (cm) Plant condition Example 1 30 28 93.3 12.5 2.7 The plant has more branches, denser, and bright green leaves, and more lateral roots. Comparative Example 1 30 13 43.3 3.2 2.0 Fewer root systems, seedlings prone to dying Comparative Example 2 30 27 90.0 4 3.5 Long root system, dark green leaves Comparative Example 3 30 27 90.0 10.3 2.1 Numerous lateral roots, relatively short roots, easily broken, and leaf edges prone to scorching during hardening-off process. Comparative Example 4 30 22 73.3 12.5 1.5 The root system has many branches, short and thick roots, and is prone to browning. Comparative Example 5 30 15 50.0 3.2 2.2 Fewer root systems, seedlings prone to dying Comparative Example 6 30 37 90.0 4.5 3.8 The plant has more branches, denser, and bright green leaves, and more lateral roots. Comparative Example 7 30 22 73.3 12.7 1.5 The plant has numerous branches, grows in clusters, has thick leaves, short and thick roots, and is prone to browning.
[0059] As can be seen from the above embodiments, the present invention provides a double-layer rooting and strengthening culture medium for quince tissue culture seedlings and a method for rooting and hardening quince tissue culture seedlings, which can improve the rooting rate and transplant survival rate of quince tissue culture seedlings.
[0060] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.
Claims
1. A double-layer rooting and seedling-strengthening culture medium for quince tissue culture seedlings, characterized in that, The upper layer of the double-layer rooting and seedling strengthening medium consists of: 1 / 2 MS + 6.8~7.2 g / L agar + 28~32 g / L sucrose + 0.8~1.2 mg / L NAA; The lower layer of the double-layer rooting and seedling strengthening medium consists of: MS + 6.8~7.2 g / L agar + 28~32 g / L sucrose + 0.4~0.6 mg / L 6-BA.
2. A method for rooting and hardening off quince tissue culture seedlings, characterized in that, Includes the following steps: S1. After 4 weeks of subculture, select robust tissue culture seedlings, remove the swollen callus tissue and leaves from the lower part of the tissue culture seedlings, and inoculate them onto the double-layer rooting and strengthening culture medium for quince tissue culture seedlings as described in claim 1 for culture. After inoculation, the cells were first cultured in the dark at 24.5~25.5℃ for 5 days, and then cultured for another 2~3 days under a light intensity of 180~220 lx and a photoperiod of 14 L / 10D. Finally, the cells were transferred to a light intensity of 1800~2200 lx and a photoperiod of 14 L / 10D. S2. After culturing for 24-26 days, open the bottle mouth of the rooted seedlings by 1 / 2 to harden the seedlings for 2-3 days, and then open the bottle mouth completely to harden the seedlings for 2-3 days. The hardening temperature is 23-27℃, the light intensity is 1800-2200 lx, and the photoperiod is 14L / 10D. S3. Remove the tissue culture seedlings from the culture medium, wash off the culture medium from the roots, then soak the roots of the tissue culture seedlings in a carbendazim solution for 28-30 seconds before transplanting them into the cultivation substrate.
3. The method according to claim 2, characterized in that, The robust tissue culture seedlings are those with a height ≥3 cm, stem diameter ≥0.3 mm, and no wilting leaves.
4. The method according to claim 2, characterized in that, The cultivation substrate comprises vermiculite, nutrient soil, and perlite in a volume ratio of 1:1:
1.
5. The method according to claim 4, characterized in that, Before transplanting tissue culture seedlings, the cultivation substrate is sterilized with carbendazim solution, wherein the volume ratio of carbendazim solution to cultivation substrate is 1:9~11.
6. The method according to claim 2 or 5, characterized in that, The carbendazim solution is a 900-1100 times diluted solution of carbendazim.
7. The method according to claim 2, characterized in that, After transplanting, cover the seedling cultivation box with a humidification chamber for 9-11 days, controlling the substrate moisture at 83%-87%, the temperature at 24.5-25.5℃, the air humidity at 48-52%, and the photoperiod at 16L / 8D. After 9-11 days, remove the humidification chamber and transplant the seedlings to the field when they reach a height of 15-20 cm.
8. The method according to claim 2, characterized in that, Before transplanting to the field, quince tissue culture seedlings are cultured in an artificial climate chamber.