Rapid propagation method for cunninghamia lanceolata by taking stem segment as explant
By using stem segments as explants and induction, proliferation and regenerating root in culture media with different formulas, the problem of single asexual reproduction mode of Luotian weeping clover is solved, and rapid reproduction and maintaining the excellent variety is achieved.
Patent Information
- Application Number
- CN202411771166.3
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-12-04
- Publication Date
- 2025-03-25
AI Technical Summary
The asexual reproduction method of Luotian weeping cypress is single, and the rapid reproduction system based on plant tissue culture technology is lacking, resulting in low reproduction efficiency and difficult to maintain the excellent variety.
The stem segment was used as an explant, and the culture medium of different formulas (MS+ agar+sucrose+6-BA+IBA, MS+ agar+sucrose+6-BA+IBA, 1/2MS+ agar+sucrose+SNP+NAA) was successfully established. The sterile seedling micro-propagation and rapid breeding system of Luotian Cypress Cyrus was successfully established.
It realizes the rapid reproduction of Luotian weeping cypress and the excellent maintenance of variety, has a high transplant survival rate, and provides a reliable asexual reproduction method.
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Figure CN119678846A_ABST
Abstract
Description
Technical Field
[0001] The invention relates to the technical field of plant tissue culture, and in particular to a rapid propagation method of a Luotian weeping fir with stem segments as explants. Background Art
[0002] Luotian weeping fir ( Cunninghamia lanceolata cv. Luotian) is a natural variant found in Luotian County, Huanggang, Hubei. Compared with the original species, its characteristics are better, with good adaptability, and can withstand water and barrenness. Its crown is narrow and spire-shaped, with a crown width of about 1.5m. The branches are short and thin, 80~100cm long and 1~2cm thick. Old branches over 2~3 years old droop, and the side branches stop growing after 3 years. The branches are slender. When the side branches grow to 6~7 years old, they fall off naturally in the autumn and winter of the same year. The remaining branches are gradually buried by the trunk, becoming a good tree without knots. It has stable genetic performance and is a good economic tree species. It can also be used as a greening tree species. Luotian weeping fir has become an ideal plant type among forest trees due to its unique morphological characteristics. It is not only of considerable economic value, but also has broad prospects for promotion and application.
[0003] The fruiting period of Luotian weeping fir is late and the fruit yield is small due to its narrow crown, making it difficult to collect seeds and the seeds are not plump. In addition, the seeds collected from the natural forest are contaminated by ordinary fir pollen, which causes the offspring to differentiate, and only a small part of them show typical characteristics of weeping fir. Therefore, asexual reproduction is the main and most important reproduction method of Luotian weeping fir.
[0004] At present, the main method of raising seedlings of Luotian weeping fir is cuttings, but this process relies on natural factors, takes a long time to raise seedlings, and long-term reproduction will lead to degeneration. Plant tissue culture is an asexual reproduction technology that uses plant tissues to obtain regenerated plants. Under a suitable culture system, it can achieve rapid reproduction and effectively maintain the quality of the variety. However, there is currently a lack of a regeneration and rapid propagation system based on plant tissue culture technology suitable for Luotian weeping fir. Summary of the invention
[0005] In view of the technical problems existing in the background technology, the present invention provides a rapid propagation method of Luotian weeping twig cedar using stem segments as explants, so as to improve the current situation that the asexual propagation mode of Luotian weeping twig cedar is single and there is a lack of regeneration and rapid propagation technology.
[0006] The technical solution of the present invention is as follows: A method for rapid propagation of Lutian weeping fir with stem segments as explants, comprising the following steps: S1. Using the stem segments of Luotian weeping fir as explants, inoculating them on the first culture medium for induction culture; S2, inoculating the adventitious buds obtained in step S1 into a second culture medium for proliferation culture; S3, inoculating the adventitious buds obtained in step S2 into a third culture medium for regenerated root induction culture; S4, transplanting the sterile seedlings obtained in step S3; Among them, the first culture medium is MS+agar+sucrose+6-benzylaminopurine (6-BA)+3-indolebutyric acid (IBA), the second culture medium is MS+agar+sucrose+6-BA+IBA, and the third culture medium is 1 / 2MS+agar+sucrose+sodium nitroprusside (SNP)+NAA (naphthaleneacetic acid).
[0007] Preferably, in the above-mentioned rapid propagation method of C. truncatum, the first culture medium is MS+sucrose+agar+0.9mg / L 6-BA+0.4mg / L IBA. The data of the embodiment show that when the first culture medium is MS+30g / L sucrose+7g / L agar+0.9mg / L 6-BA+0.4mg / L IBA, it is best to induce adventitious buds in the stem segments of C. truncatum, and has the advantages of high induction rate, no browning, and strong seedlings.
[0008] More preferably, under the above-mentioned first culture medium conditions, the culturing time in step S1 is 30 days.
[0009] Preferably, in the above-mentioned rapid propagation method of C. truncatula, the second culture medium is MS+sucrose+agar+0.9mg / L 6-BA+0.4mg / L IBA. The data of the embodiment show that when the second culture medium is MS+30g / L sucrose+7g / L agar+0.9mg / L 6-BA+0.4mg / L IBA, it is most suitable for adventitious bud proliferation, and the proliferation coefficient can reach 5.66.
[0010] More preferably, under the above-mentioned second culture medium conditions, the culturing time in step S2 is 30 days.
[0011] Preferably, in the above-mentioned rapid propagation method of Luotian weeping cypress, the third culture medium is 1 / 2MS+agar+sucrose+9mg / LSNP+0.1mg / L NAA. The data of the embodiment show that SNP, as an exogenous NO donor, can induce the occurrence of adventitious roots of Luotian weeping cypress, and the formation of adventitious roots is dose-dependent. When the concentration of SNP reaches 9mg / L, the roots of Luotian weeping cypress grow well; SNP and an appropriate amount of NAA can significantly improve the rooting effect of Luotian weeping cypress, and the survival rate of the resulting seedlings after transplantation is extremely high and the growth is excellent.
[0012] More preferably, under the above-mentioned third culture medium conditions, the culture conditions in step S3 are: light intensity 2000 Lux, daily illumination 16 h, temperature 23±1° C., and culture 60 d.
[0013] Preferably, in the above-mentioned Luotian weeping twig rapid propagation method, after transplanting in step S4, the plants are covered with a sunshade net and treated with carbendazim regularly.
[0014] In the above-mentioned rapid propagation method of Luotian weeping twig cedar, the stem segments of Luotian weeping twig cedar need to be disinfected before inoculation, and the treatment method is preferably: first soak in 3% sodium hypochlorite solution for 8-15 minutes, rinse with sterile water and then treat with 75% alcohol for 20-60 seconds, rinse with sterile water and then absorb the moisture.
[0015] Preferably, in the above-mentioned rapid propagation method of Cryptomeria japonica, the pH of the first culture medium, the second culture medium and the third culture medium are all 5.8±0.5.
[0016] Compared with the prior art, the present invention has the following beneficial effects: In the construction of plant regeneration and rapid propagation systems, plant growth regulators play an important role. However, existing studies have also shown that for different plants, the type and content of regulators are key factors affecting the rapid propagation system, and there is still a technical gap in the rapid propagation system of Luotian weeping cypress. The present invention uses the tender stems of Luotian weeping cypress as explants, and through the study of adventitious bud induction, proliferation culture and adventitious root induction using growth regulators, successfully establishes for the first time a sterile seedling micropropagation and seedling rapid propagation system of Luotian weeping cypress, and has an extremely high transplant survival rate, providing a reliable way to retain the excellent traits of Luotian weeping cypress and rapid propagation. BRIEF DESCRIPTION OF THE DRAWINGS
[0017] In order to more clearly illustrate the technical solution of the present invention, the following is a brief introduction to the drawings used in the present invention. Obviously, the drawings described below are only some embodiments of the present invention, and for those skilled in the art, other drawings can be obtained based on these drawings without creative work.
[0018] Figure 1 It is a schematic diagram of the induction of adventitious buds of Pseudotsuga truncatula under different treatments in the embodiments of the present invention; Figure 2 This is a comparison diagram of the adventitious bud growth of Cunninghamia lanceolata under different treatments in the embodiments of the present invention; Figure 3 This is a schematic diagram of the proliferation of adventitious buds of Pseudotsuga truncatula under different treatments in the embodiments of the present invention; Figure 4 This is a comparison of the adventitious root morphology of Cryptomeria japonica under different treatments in the examples of the present invention. DETAILED DESCRIPTION
[0019] The following embodiments of the technical solution of the present invention are described in detail in conjunction with the accompanying drawings. The following embodiments are only used to more clearly illustrate the technical solution of the present invention, and are therefore only used as examples, and cannot be used to limit the protection scope of the present invention.
[0020] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by technicians in the technical field to which the present invention belongs; the terms "including" and "having" in the specification and claims of the present invention and any modifications thereof are intended to cover non-exclusive inclusions; the terms "first", "second", etc. in the specification and claims of the present invention are only used to distinguish different objects and cannot be understood as indicating or implying relative importance or implicitly indicating the primary and secondary relationship of the indicated technical features.
[0021] Some specific embodiments are listed below. It should be noted that the embodiments described below are exemplary and are only used to explain the present invention, and should not be understood as limiting the present invention.
[0022] The pretreatment method of the explants used in the following examples is as follows: the leaves of the tender stems of the weeping fir were removed and washed thoroughly with detergent, and then repeatedly rinsed with tap water for 2 hours, and a 1.5 cm long stem segment with buds was cut from the rinsed tender stems of the weeping fir as an explant, and the explant was treated with 3% sodium hypochlorite solution on a clean bench for 10 minutes, and was constantly stirred with a glass rod during the treatment process. After being rinsed with sterile water for 2-3 times, it was treated with 75% alcohol for 30 seconds, and then rinsed with sterile water for 4-5 times, and the treated explant was placed on sterile filter paper to absorb the moisture for inoculation.
[0023] The preparation method of the culture medium used in the following examples is as follows: according to the specific composition of different culture media, weigh an appropriate amount of MS medicine and sucrose and put them into a large beaker filled with distilled water, then stir with a glass rod until completely dissolved, and dispense the completely dissolved solution into conical flasks of suitable sizes, add corresponding plant growth regulators, agar and other substances; adjust the pH value of the culture medium in each conical flask to 5.8 by pH test paper, and then sterilize it in an autoclave, and the program of the autoclave is set to: pressure 0.105Mpa, temperature 121°C, sterilization 30min. After sterilization, dispense the culture medium into tissue culture bottles in a clean bench. After the dispensing is completed and cooled and solidified, transfer it to the observation room and observe for three days. If the culture medium is not contaminated, it can be used for inoculation.
[0024] If no specific techniques or conditions are specified in the examples, the techniques or conditions described in the literature in the field or the product instructions are used. If no manufacturer is specified for the reagents or instruments used, they are all conventional products that can be purchased commercially.
[0025] Example 1 This example provides a method for rapid propagation of Luotian weeping fir, comprising the following steps: (1) The disinfected stem segments of P. truncatula (about 1.5 cm) were inoculated on a culture medium containing MS + 30 g / L sucrose + 7 g / L agar + 0.9 mg / L 6-BA + 0.4 mg / L IBA and cultured for 30 days. The culture conditions were: light intensity 2000 Lux, daily light exposure 16 h, and temperature 23 ± 1 °C.
[0026] (2) The adventitious buds obtained in step (1) were inoculated into a culture medium for proliferation culture, and cultured under the conditions of a light intensity of 2000 Lux, 16 h of light per day, and 23±1°C for 30 days. The culture medium used was specifically MS+30 g / L sucrose+7 g / L agar+0.9 mg / L 6-BA+0.4 mg / L IBA.
[0027] (3) Inoculate the adventitious buds of C. truncatula of about 1.5 cm into the rooting medium. After inoculation, place the tissue culture bottle in the culture room and culture for 60 days under the conditions of 2000 Lux light intensity, 16 h of light per day, and 23±1°C. The rooting medium is 1 / 2MS+30g / L sucrose+7g / L agar+0.1mg / L NAA+9mg / L SNP.
[0028] (4) Transplant the seedlings of Pseudotsuga truncatula obtained in step (3) into a greenhouse, cover them with a shade net, and water them with 1 g / L carbendazim every three days. Calculate the survival rate after 30 days.
[0029] Example 2 Different from Example 1, the contents of 6-BA and IBA in the culture medium used in step (1) were adjusted in this example, and the treatments are shown in Table 1.
[0030] The induction rate, contamination rate, browning rate, earliest germination time and adventitious bud length of the adventitious buds in the process of step (1) were respectively counted in Example 1 and the present example, a total of 16 treatments. Specifically, 6 bottles were treated (one stem segment with buds was inoculated in each bottle), and the average value was measured three times. The test results are shown in Tables 1 and 2.
[0031] Table 1 Comparison of adventitious bud induction under different treatments
[0032] Note: Different lowercase letters indicate significant differences ( P ≤0.05).
[0033] As shown in Table 1, under the culture medium conditions of Example 1, buds can be seen growing on the stem segments of the Luotian weeping fir as early as the 9th day (see Figure 1A). Except for the medium without 6-BA and IBA, adventitious buds can be induced under other culture treatment conditions, indicating that adding 6-BA and IBA to MS medium can effectively promote the induction of adventitious buds in the stem segments of Luotian weeping fir. However, it should be noted that the induction effect is significantly different under different treatments. For example, under the conditions of Example 1, the induction rate of adventitious buds of Luotian weeping fir is as high as 100%, and there is no browning phenomenon, while under other conditions, there is browning phenomenon (see Figure 1 B, specifically, the plants in treatment group 3 turned brown and died on the 13th day of culture, with the highest browning rate reaching 20%, and the lowest induction rate being as low as 20%.
[0034] Table 2 Comparison of adventitious bud growth of Luotian cypress under different treatments
[0035] Note: Different lowercase letters indicate significant differences ( P ≤0.05).
[0036] As shown in Table 2, in treatment 9, the number of adventitious buds induced by the stem segments of Luotian truncatula was the largest (see Figure 2 A), reaching 5.08; secondly, in treatments 10 and 14, the number of adventitious buds induced by the stem segments of Luotian weeping fir reached 4.38. However, although there were many buds under the above treatment conditions, they grew slowly or were weak (see Figure 2 B and 2C); Under the conditions of Example 1 of the present invention, the average number of adventitious buds induced by the stem segments of Luotian weeping fir was 4.33, which was not the largest, but the bud length was the longest, at 1.62 cm, and it grew fast and had strong seedlings (see Figure 2 D).
[0037] Example 3 Different from Example 1, the contents of 6-BA and IBA in the culture medium used in step (2) were adjusted in this example, and the treatments are shown in Table 3.
[0038] The proliferation coefficient and browning rate of step (2) in the 16 treatments in Example 1 and this example were respectively counted. Specifically, 4 bottles were treated (3 adventitious buds were inoculated in each bottle), and the average value was measured three times. The test results are shown in Tables 3 and Figure 3 (A is treatment 16, B is treatment 15).
[0039] Table 3 Comparison of the proliferation of adventitious buds of Cunninghamia lanceolata under different treatments
[0040] Note: Different lowercase letters indicate significant differences ( P ≤0.05).
[0041] From the above results, it can be determined that the optimal culture medium for the proliferation of adventitious buds in the stem segments of Luotian weeping fir is MS + 0.9mg / L 6-BA + 0.4mg / L IBA + 30g / L sucrose + 7g / L agar, and the proliferation coefficient can reach 5.66.
[0042] Example 4 The difference from Example 1 is that the content of SNP, NAA and IBA in the culture medium used in step (3) was adjusted in this example. There are 16 treatments in Example 1 and this example, and each treatment is shown in Table 4, where treatment 15 is Example 1.
[0043] Table 4 Composition of culture medium under different treatments
[0044] The rooting rate, root length, number of rooted branches and other parameters of the Luotian weeping fir obtained in step (3) in the above 16 treatments were counted respectively. Specifically, 6 bottles were inoculated in each treatment (3 stem tips were inoculated in each bottle), and the average value was measured three times. The test results are shown in Tables 5 and Figure 4 shown.
[0045] Table 5 Statistics on rooting of C. truncatula under different treatments
[0046] Note: Different lowercase letters indicate significant differences ( P ≤0.05).
[0047] Figure 4 The following are photos of adventitious roots of P. truncatum under some treatments, among which A is treatment 6, B is treatment 8, C is treatment 9, and D is treatment 12. From the morphological comparison of adventitious roots of P. truncatum under different treatments, it can be seen that the base of P. truncatum is brown and the leaves are yellow in treatments 2, 7, 10, and 11, the leaves are lush and the tillers are more in treatments 6, 8, and 12, the roots in treatment 6 are the most and the longest, and the roots in treatment 5 are the shortest. At the same time, it can be seen from Table 5 that NO donors have a dose-dependent effect on the formation of adventitious roots. When the SNP concentration reaches 9 mg / L, the growth of plant roots is better, and the root system of plants in treatment 6 has better growth and development. From treatments 1-4, it can be seen that plants can take root normally when IBA and NAA are added to the culture medium, but the growth condition is not good. Among the above treatments, treatment 6 had the best rooting effect, with a rooting rate (76.66%), average root length (24.25 cm), average number of roots (5), and root surface area (9.17 cm 2 ) are the highest.
[0048] Under the different treatment conditions shown in Table 4, the growth of the weeping fir after transplanting in step (4) was further statistically analyzed, and the results are shown in Table 6.
[0049] Table 6 Statistics of the growth of Luotian weeping fir after transplantation under different treatments
[0050] Note: Different lowercase letters indicate significant differences ( P ≤0.05).
[0051] As shown in Table 6, the survival rates of treatments 5, 6, and 12 were significantly higher than those of the other treatment groups, and the survival rate of treatment 13 was the lowest; the number of new branches in treatments 6, 7, 14, and 16 was significantly higher than that in the other treatment groups, among which treatment group 6 had the largest number of new branches, 10.33, and treatment 12 had the least number of new branches, 1.33.
[0052] In summary, the present invention successfully obtains a sterile seedling micropropagation and rapid propagation system for the weeping twig larvae, which is of great significance to the reproduction of the weeping twig larvae.
[0053] It should be noted that the present invention is not limited to the above-mentioned embodiments. The above-mentioned embodiments are only examples, and the embodiments having the same structure as the technical idea and exerting the same effect within the scope of the technical solution of the present invention are all included in the technical scope of the present invention. In addition, without departing from the scope of the main purpose of the present invention, various modifications that can be thought of by those skilled in the art to the embodiments and other methods of combining some of the constituent elements in the embodiments are also included in the scope of the present invention.
Claims
1. A method for rapid propagation of Cryptomeria japonica L. using stem segments as explants, characterized in that: The following steps are involved: S1. Using the stem segments of Luotian weeping fir as explants, inoculating them on the first culture medium for induction culture; S2, inoculating the adventitious buds obtained in step S1 into a second culture medium for proliferation culture; S3, inoculating the adventitious buds obtained in step S2 into a third culture medium for regenerated root induction culture; S4, transplanting the sterile seedlings obtained in step S3; Among them, the first culture medium is MS+agar+sucrose+6-BA+IBA, the second culture medium is MS+agar+sucrose+6-BA+IBA, and the third culture medium is 1 / 2MS+agar+sucrose+SNP+NAA.
2. The rapid propagation method of Cunninghamia lanceolata according to claim 1, characterized in that: The first culture medium is MS+sucrose+agar+0.9 mg / L 6-BA+0.4 mg / L IBA.
3. The rapid propagation method of Cunninghamia lanceolata according to claim 2, characterized in that: The culturing time in step S1 is 30 days.
4. The rapid propagation method of Cunninghamia lanceolata according to claim 1, characterized in that: The second culture medium is MS+sucrose+agar+0.9 mg / L 6-BA+0.4 mg / L IBA.
5. The rapid propagation method of Cunninghamia lanceolata according to claim 4, characterized in that: The culturing time in step S2 is 30 days.
6. The method for rapid propagation of Cunninghamia lanceolata according to claim 1, characterized in that: The third culture medium is 1 / 2MS+agar+sucrose+9mg / L SNP+0.1mg / L NAA.
7. The method for rapid propagation of Cunninghamia lanceolata according to claim 6, characterized in that: The culture conditions in step S3 are: light intensity 2000 Lux, daily light exposure 16 h, temperature 23 ± 1 ° C, and culture for 60 days.
8. The rapid propagation method of Cunninghamia lanceolata according to claim 1, characterized in that: After transplanting in step S4, the plants are covered with a sunshade net and treated with carbendazim.
9. The rapid propagation method of Cunninghamia lanceolata according to claim 1, characterized in that: In step S1, the stem segments are disinfected before inoculation, and the disinfection method is: first soak in 3% sodium hypochlorite solution for 8-15 minutes, rinse with sterile water, then treat with 75% alcohol for 20-60 seconds, rinse with sterile water and then absorb the moisture.
10. The rapid propagation method of Cunninghamia lanceolata according to claim 1, characterized in that: The pH of the first culture medium, the second culture medium and the third culture medium are all 5.8±0.5.