Method for rapidly germinating and raising seedlings of ilex chinensis seeds

Abstract

The invention discloses a method for rapid germination and seedling raising of holly seeds. The method comprises the following steps: rubbing and cleaning holly fruits in a color changing period to obtain seeds; disinfecting and airing the cleaned seeds, cutting the seeds into two halves, and inoculating half of the seeds with embryos into a seed germination culture medium; cutting the cultured seedlings into stem segments with axillary buds; inoculating the stem segments to an axillary bud proliferation culture medium for culturing; culturing until small buds are differentiated from the axillary buds, improving the illumination intensity, and when the differentiated small seedlings grow to 2 cm or above, cutting off the small seedlings and transferring the small seedlings to a strong seedling culture medium for culturing; and after strong seedling culture, cutting a single bud seedling which grows robustly and transferring to a rooting culture medium for culture. According to the method, only several months are needed from seed treatment to germination to rooting and seedling formation, and tedious steps such as variable-temperature sand storage are not needed. Seed shell breaking in the color changing period is taken as a core starting point, tissue culture hormone is coupled for precise regulation and control, chain type efficient conversion of germination-proliferation-rooting is realized, and the seedling raising period is shortened by 60% or above.

Description

Technical Field

[0001] The invention belongs to the technical field of garden plant cultivation and seed and seedling cultivation, and more specifically relates to a method for rapidly germinating and growing seedlings from holly seeds. Background Art

[0002] Holly (Ilex chinensis Sims) breeding has been slow to start and progress has been slow, with a lack of new varieties and improved forest tree species. Besides the lack of advanced breeding techniques, one of the limiting factors in holly breeding is the natural three-year dormancy of holly seeds. Studies have shown that the germination rate of first-year seeds is zero, less than 10% after one year of storage at room temperature, and less than 20% after one year of storage at 4°C (Huang Hairong, Effects of Storage Methods on Allelochemical Composition and Seed Germination Rate of Two Holly Seeds). Compared to seeds of the same genus, Ilex chinensis Sims, which had been stored in sand for one year, the germination rate of the former was zero, while the germination rate of the latter averaged 27% (Yu Youxiang et al., Observations on Seed Germination and Seedling Stage of 'Oster' Ilex chinensis Sims). After treating Ilex macrocarpa seeds with concentrated sulfuric acid for 1.5 hours and then stratifying them with a 250mg·L-1 GA3 + 0.2% KNO3 solution for one winter, the germination rate can reach 17.1% (Xu Benmei et al., "A Preliminary Study on Dormancy and Germination of Ilex macrocarpa Seeds"). Ilex macrocarpa seeds are often stored in wet sand at varying temperatures for 1-1.5 years before sowing. They are then soaked in 40°C warm water for 12 hours, stratified at 5°C for 24 hours, and then soaked again in 40°C warm water for 10 hours. After disinfection and germination in a sand bed for three months, the germination rate can reach 30%, shortening the dormancy period by 9-12 months (Deng Xiuyan et al., "Cultivation Technology of Ilex macrocarpa"). A seed treatment method for holly (Ilex purpurea) involves soaking the seeds in 98% concentrated sulfuric acid for 5 minutes, then soaking them in a 500 mg·L⁻¹ GA⁻¹ solution for 48 hours, followed by warm stratification for 60 days, and then cold stratification for 160 days. The germination rate after treatment reached 51% (Wei Chaoyang, "Mechanism of Dormancy and Method for Relieving Dormancy in Holly Seeds"). This indicates that existing techniques for germinating holly seeds, as well as most other dormant Ilex species, require a long time and complex procedures. K. Yao & M.B. Deng, also from the Dabieshan holly (Ilex dabieshanensis), collected berries and excised a portion of the seed coat under a dissecting microscope until the embryo was exposed. Subsequently, sterile seedlings were obtained within the same year using axillary bud initiation medium, adventitious bud rooting medium, and rooting medium (Zhou Yanwei et al., "A Rapid Propagation Method for Dabieshan Holly"). However, this document does not describe the germination rate of embryos under sterile conditions, nor does it include orthogonal design screening of subsequent hormone combinations. Summary of the Invention

[0003] The technical problem to be solved by the present invention is at least how to shorten the holly seedling raising period and improve the germination rate, and provide a method for quickly germinating and growing holly seeds into seedlings.

[0004] The present invention provides a method for rapidly germinating and growing holly seeds into seedlings, comprising:

[0005] The holly fruits at the color change stage are rubbed and cleaned to obtain seeds; after cleaning, the seeds are disinfected and dried;

[0006] Cut the dried seeds into two halves, and take the half with embryo to inoculate the seed germination medium;

[0007] After the holly seeds germinate, the culture medium is replaced every month until the seedlings grow to 5-7 cm tall; the cultured seedlings are cut into 1.5-2.5 cm stem segments with axillary buds;

[0008] The stem segments were inoculated onto axillary bud proliferation medium for culture;

[0009] Cultivate until the axillary buds differentiate into small buds, increase the light intensity, and wait until the differentiated seedlings grow to more than 2 cm in height, cut the seedlings and transfer them to the seedling medium for cultivation;

[0010] After the seedlings are cultured, single healthy seedlings are cut and transferred to the rooting medium for culture.

[0011] Preferably, the seed germination medium consists of MS medium, 1 mg / mL GA3, and 0.5 g activated carbon.

[0012] Preferably, the axillary bud proliferation culture medium consists of MS culture medium, 0.05 mg / mL NAA, 0.5 mg / mL Zeatin, 1 mg / mL GA3, and 0.5 g activated carbon.

[0013] Preferably, the seedling-strengthening culture medium consists of MS culture medium + 0.05 mg / mL NAA, 0.2 mg / mL Zeatin, and 0.5 g activated carbon.

[0014] Preferably, the rooting medium consists of 1 / 2 MS medium, 0.05 mg / mL NAA, 0.1 mg / mL IBA, and 0.5 g activated carbon.

[0015] Preferably, the culture temperature is 24° C.-26° C., and the light culture conditions include a light intensity of 1000-2500 lux and a light duration of 12 hours per day.

[0016] Preferably, the color change period is when the fruit skin color is between green and red.

[0017] Preferably, the color change period is 6-8 weeks after the cessation of expansion.

[0018] The beneficial effects of the present invention are:

[0019] The present invention selects the physiological stage of 6-8 weeks after the fruit stops expanding and the epidermis turns from green to red as the explant. At this time, the seed embryo is fully developed but the inhibitory substance has not reached its peak. By cutting the seeds in half to expose the embryo, the physical barrier of the seed coat and the germination inhibitor are directly broken, and the germination rate of the seeds cut in half during the color change period is improved in coordination with the tissue culture environment.

[0020] Compared to traditional methods, this method takes only 4-5 months from seed preparation to germination to rooting and seedling formation, eliminating the need for cumbersome steps like temperature-controlled sand storage. By focusing on seed shelling during the color change period and coupling it with precise control of tissue culture hormones, this method achieves a highly efficient, chain-like transformation from germination to proliferation to rooting, shortening the seedling cultivation cycle by over 60%. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 : Germination of holly seeds at different stages, including (A) holly fruits and seeds at F1-F6 stages, scale is 1 cm; (B) germination of seeds at different stages after being cut in half or directly sown on MS basal medium.

[0022] Figure 2 : Rapid propagation of holly sterile seedlings through tissue culture, including (A) seed germination seedlings; (B) seedling culture; (C) rooting culture, scale is 1 cm.

[0023] Figure 3 : Ilex tissue culture seedlings cultured in substrate, the scale is 2 cm. DETAILED DESCRIPTION

[0024] To make the objectives, technical solutions, and advantages of the embodiments of the present invention more clear, the technical solutions of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the embodiments described are only part of the embodiments of the present invention, not all of them. All other embodiments obtained by ordinary technicians in this field based on the embodiments of the present invention without making any creative efforts shall fall within the scope of protection of the present invention.

[0025] The existing work of holly (Ilex chinensis Sims) breeding has a long seedling raising cycle and a low germination rate. Therefore, there is at least one problem to be solved: how to shorten the holly seedling raising cycle and improve the germination rate.

[0026] Based on this, the embodiment of the present invention provides a method for rapidly germinating and growing seedlings from holly seeds, which mainly adopts tissue culture, selects seeds from a specific period, and breaks the seed coat, thereby improving the germination rate and shortening the breeding cycle. Specifically, the method includes:

[0027] The fresh holly berries are rubbed and cleaned to obtain seeds. For example, the seeds can be cleaned by the following steps: washing with detergent for 3 minutes, washing with tap water for 3 minutes, and rinsing with running water for 10 minutes.

[0028] After the cleaning treatment, the seeds are sterilized. As an example, the seeds can be sterilized by the following steps: 75% alcohol for 1 minute, 3 times with sterile water, 30% sodium hypochlorite for 25 minutes, and 5 times with sterile water.

[0029] After sterilization, the seeds are placed on sterile filter paper and allowed to air dry briefly. The air-dried seeds are then cut in half, and the embryo-bearing half is inoculated onto a seed germination medium. The culture temperature is approximately 25°C, the light intensity is 1000-2000 lux, and the illumination time is 12 hours per day. For example, the seed germination medium is MS medium supplemented with 1 mg / mL gibberellin (GA3) and 0.5 g activated carbon.

[0030] After the holly seeds germinate, the seed germination medium should be replaced every month. The culture temperature should be around 25℃, the light intensity should be 1000-2000 lux, and the light time should be 12 hours / day. The seeds should grow to about 7cm in height. Figure 2 In (A), the cultured seedlings were cut into approximately 2 cm stem segments with axillary buds and inoculated onto axillary bud proliferation medium. The culture temperature was approximately 25°C, the light intensity was 1000-2000 lux, and the illumination time was 12 hours / day. As an example, the axillary bud proliferation medium used was MS medium + 0.05 mg / mL naphthaleneacetic acid NAA + 0.5 mg / mL zeatin + 1 mg / mL gibberellin A3 (GA3) + 0.5 g activated carbon.

[0031] The culture medium is incubated until axillary buds differentiate into small buds at a temperature of approximately 25°C, with a light intensity of 1500-2500 lux for 12 hours per day. The differentiated seedlings grow to a height of more than 2 cm, are cut and transferred to a seedling medium for incubation at a temperature of approximately 25°C, with a light intensity of 1000-2000 lux for 12 hours per day. For example, the seedling medium comprises MS medium + 0.05 mg / mL NAA + 0.2 mg / mL zeatin + 0.5 g activated carbon.

[0032] After the seedlings are grown, see Figure 2 In (B), a single healthy sprout was cut and transferred to a rooting medium for cultivation at a temperature of about 25°C, a light intensity of 1000-2000 lux, and a light duration of 12 hours per day. Figure 2 In the medium (C), the rooting rate reached 92.59%. As an example, the rooting medium used was 1 / 2 MS medium + 0.05 mg / mL NAA + 0.1 mg / mL indolebutyric acid IBA + 0.5 g activated carbon.

[0033] The results of holly tissue culture seedlings cultured in substrate are shown in Figure 3 .

[0034] Test Example 1

[0035] This experiment was used to determine the effect of different picking times on seed germination of holly. Sampling was carried out every two weeks from the time the fruit stopped expanding to the time the fruit was fully mature. Sampling was stopped when the fruit was fully mature and turned red. A total of 6 samplings were conducted over a period of 3 months. The seed samples were recorded as F1, F2, F3, F4, F5 and F6. Figure 1 Middle (A). F1 is when the fruit stops expanding, F4 is when it just reaches the color change stage, and F6 is when the fruit is fully mature and turns red.

[0036] Test Example 2

[0037] This experiment was used to determine the effect of seed coat integrity on seed germination. The cleaned seeds were sown as whole seeds or half seeds. The germination results of the two different sowing methods on MS basal medium were compared. Figure 1 Middle (B). HS means half-seed sowing, and WS means whole seed sowing.

[0038] Test Example 3

[0039] To determine the effect of gibberellin A3 (GA3) on seed germination, sterilized F4 stage whole seeds and halved seeds were sown on MS medium, and different concentrations of GA3 were added: 0 mg / L, 0.5 mg / L, 1 mg / L, 1.5 mg / L, and 2 mg / L.

[0040] Table 1 Effects of different GA3 concentrations and seed morphology on seed germination

[0041]

[0042] Table 1

[0043]

[0044]

[0045] The results of a three-factor experiment showed that there were interactions between seed morphology, sampling time, and GA3 treatment. Seed morphology had the greatest impact on the germination rate of holly seeds, followed by sampling time. Treatment with an appropriate concentration of GA3 also promoted germination. Furthermore, according to experimental results, when holly seeds were harvested at the F4 stage, halved, and treated with 1 mg / mL of GA3, the germination rate reached 65.4%.

[0046] Test Example 4

[0047] To determine the effect of different hormone ratios on axillary bud proliferation, the cultured seedlings were cut into stem segments with axillary buds of about 2 cm and inoculated into axillary bud proliferation medium: MS medium + NAA + Zeatin + GA3 + 0.5 g activated carbon. The hormone formula in the medium was L9 (3 4 ) design (Table 2), and the average height of the axillary buds of 30 seedlings counted over 60 days was used as the evaluation indicator.

[0048] Table 2 Hormone ratio of holly axillary bud proliferation culture medium L9 (3 4 ) Orthogonal experimental factors and levels

[0049]

[0050] As shown in Table 3, through orthogonal experiments and calculated R values, the factors influencing holly axillary bud proliferation, from greatest to least, are C > A > B, that is, GA3 > NAA > Zeatin. The optimal hormone ratio was A2B1C2, resulting in an NAA concentration of 0.05 mg / L, a Zeatin concentration of 0.5 mg / L, and a GA3 concentration of 1 mg / L. Under these conditions, axillary buds reached a height of 7.22 cm after 60 days, accelerating the proliferation of tissue culture seedlings.

[0051] Table 3 Analysis of orthogonal test results of hormone ratios in holly axillary bud proliferation culture medium

[0052]

[0053] The above is a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications are also considered to be within the scope of protection of the present invention.

Claims

1. A method for rapidly germinating holly seeds into seedlings, characterized in that: The method comprises: The holly fruits at the color change stage are rubbed and cleaned to obtain seeds; after cleaning, the seeds are disinfected and dried; Cut the dried seeds into two halves, and take the half with embryo to inoculate the seed germination medium; After the holly seeds germinate, the culture medium is replaced every month until the seedlings grow to 5-7 cm tall; the cultured seedlings are cut into 1.5-2.5 cm stem segments with axillary buds; The stem segments were inoculated onto axillary bud proliferation medium for culture; Cultivate until the axillary buds differentiate into small buds, increase the light intensity, and wait until the differentiated seedlings grow to more than 2 cm in height, cut the seedlings and transfer them to the seedling medium for cultivation; After the seedlings are cultured, single healthy seedlings are cut and transferred to the rooting medium for culture.

2. The method according to claim 1, characterized in that The seed germination medium consists of MS medium, 1 mg / mL GA3, and 0.5 g activated carbon.

3. The method according to claim 1, characterized in that The axillary bud proliferation medium consists of MS medium, 0.05 mg / mL NAA, 0.5 mg / mL Zeatin, 1 mg / mL GA3, and 0.5 g activated carbon.

4. The method according to claim 1, characterized in that The seedling-strengthening culture medium consists of MS culture medium + 0.05 mg / mL NAA, 0.2 mg / mL Zeatin, and 0.5 g activated carbon.

5. The method according to claim 1, characterized in that: The rooting medium consists of 1 / 2 MS medium, 0.05 mg / mL NAA, 0.1 mg / mL IBA, and 0.5 g activated carbon.

6. The method according to claim 1, characterized in that The culture temperature is 24°C-26°C, and the light culture conditions include a light intensity of 1000-2500 lux and a light duration of 12 hours / day.

7. The method according to claim 1, characterized in that: The color change period is when the color of the fruit skin changes from green to red.

8. The method according to claim 1 or 7, characterized in that: The color change period is 6-8 weeks after the expansion stops.

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