Establishment method of efficient regeneration system of cudrania tricuspidata stems

Abstract

The invention discloses a method for establishing a cudrania tricuspidata stem efficient regeneration system, and belongs to the technical field of plant tissue culture. The establishment method comprises the following steps: cutting healthy Cudrania tricuspidata current-year twigs into stem segments, reserving a plump bud on each stem segment, carrying out preliminary disinfection, soaking in a disinfectant, carrying out ultrasonic treatment and disinfection, using the disinfected bud as an explant, and carrying out bud induction culture, cluster bud enrichment culture and rooting culture to obtain Cudrania tricuspidata in-vitro regenerated seedlings. By screening the components of the culture medium in each stage and optimizing the in-vitro rapid propagation process of the cudrania tricuspidata, the propagation efficiency of a rapid propagation system is improved, a large number of high-quality seedlings can be produced within a short period of time, the market demand is met, and rapid development of industrialization of the cudrania tricuspidata is promoted.

Description

Technical Field

[0001] The invention relates to the technical field of plant tissue culture, in particular to a method for establishing a high-efficiency regeneration system of Cudrania tricuspidata stem segments. Background Art

[0002] Cudrania tricuspidata Carrière is a deciduous tree species of the genus Maclura in the family Moraceae. It is widely distributed in the provinces and regions of North my country, East China, Central South, and Southwest China. Cudrania tricuspidata Carrière likes light, tolerates shade and cold, and tolerates drought and barrenness. It can grow on hills, barren mountains, and embankment slopes. Cudrania tricuspidata Carrière has a wide range of uses. The tree itself can be used for landscaping and slope protection. It not only has excellent ecological adaptability, but also plays an important role in soil improvement, windbreak and sand fixation. The roots, bark, stems, leaves, and fruits of Cudrania tricuspidata Carrière can be used as medicine. The stem bark fiber can be used to make paper, the young leaves can be used to raise young silkworms, the wood core is used as a yellow dye, and the fruit can be eaten raw or used to make wine.

[0003] At present, the main propagation methods of Cudrania zedoaria are grafting and cuttings. Although grafting or cuttings can maintain the excellent characteristics of the original plant, the propagation coefficient is low and cannot meet the large supply demand. Grafting requires the provision of rootstocks, which has high labor costs. At the same time, the grafted seedlings have weak wind resistance and are easy to break. Cuttings require the selection of a large number of high-quality branches, and the propagation efficiency is low, requiring post-management, etc. The use of plant tissue culture rapid propagation technology can obtain a large number of excellent asexual seedlings in a short period of time. The plant traits are stable and the growth is neat, which can meet the demand for high-quality seedlings. There are few studies on the establishment of an efficient regeneration system for Cudrania zedoaria using tissue culture technology. Even if the regeneration system is established by referring to the currently reported Cudrania zedoaria tissue culture technology, it is still difficult to overcome the problems of low rooting rate and underdeveloped root system caused by the formation of callus tissue on the wound surface during tissue culture, which in turn leads to low quality of regenerated seedlings.

[0004] Therefore, improving the tissue culture technology of Cudrania trifoliata, optimizing the process of in vitro rapid propagation of Cudrania trifoliata, and increasing the conversion rate of the rapid propagation system will help produce a large number of high-quality seedlings in a short period of time and carry out large-scale production to meet market demand and promote the rapid development of Cudrania trifoliata industrialization. Summary of the invention

[0005] The purpose of the present invention is to provide a method for establishing an efficient regeneration system of Cudrania tricuspidata stem segments to solve the problems existing in the above-mentioned prior art. The method for establishing a Cudrania tricuspidata tissue culture regeneration system of the present invention is simple to operate, has a low pollution rate, a high survival rate, and a short culture cycle. At the same time, it improves the reproduction efficiency of the rapid propagation system, helps to produce a large number of high-quality seedlings in a short period of time, meets market demand, and promotes the rapid development of Cudrania tricuspidata industrialization.

[0006] To achieve the above object, the present invention provides the following solutions:

[0007] The present invention provides a method for establishing a high-efficiency regeneration system of Cudrania tricuspidata stem segments, comprising the following steps:

[0008] Healthy young branches of Cudrania zedoaria tree of the current year were cut into stem segments, each of which had a full bud, and after preliminary disinfection, they were immersed in disinfectant and ultrasonically treated for disinfection before being used as explants;

[0009] Inoculating the explants into a bud induction medium for bud induction culture to obtain young buds of sterile seedlings;

[0010] Cutting the tender shoots of the sterile seedlings, inoculating them into a clustered shoot proliferation medium for proliferation culture, and obtaining the proliferated clustered shoots;

[0011] cutting off the proliferated clustered buds, transferring them to a rooting medium for rooting culture, and obtaining in vitro regenerated seedlings of Cudrania tricuspidata;

[0012] The components of the bud induction medium are MS+0.5mg / L 6-BA+0.01mg / L IBA+30g / L sucrose+7g / L agar;

[0013] The components of the clustered bud proliferation medium are MS+2.0mg / L 6-BA+0.3mg / L IBA+25g / L sucrose+8g / L agar;

[0014] The components of the rooting medium are 1 / 2MS+0.5mg / L 6-BA+0.5mg / L IBA+0.05mg / L NAA+25g / L sucrose+8g / L agar.

[0015] Furthermore, the length of the stem segment is 1.5 to 2.0 cm.

[0016] Furthermore, the ultrasonic treatment time is 9 minutes.

[0017] Furthermore, the preliminary disinfection includes flushing with tap water, soaking in carbendazim solution, soaking in 84 disinfectant and soaking in alcohol solution in sequence.

[0018] Furthermore, the disinfectant is a mercuric chloride solution, and the mercuric chloride solution contains Tween 80.

[0019] Furthermore, in the rooting culture stage, the clustered buds cut are buds with a stem length of 1.0 to 1.5 cm.

[0020] Furthermore, the pH of the bud induction medium, the clustered bud proliferation medium and the rooting medium are all 5.8.

[0021] Furthermore, the culture conditions for the bud induction culture, the proliferation culture and the rooting culture are all 2800 Lux of light intensity, 25±2° C. of temperature and 16 h / d of photoperiod.

[0022] The present invention discloses the following technical effects:

[0023] The sampling site of the explant of the present invention is young branches, and the surface of the young branches of Cudrania tricuspidata is covered with short hairs. During the disinfection process, the explant and the disinfectant are in a state of ultrasonic co-oscillation, which can make the plant material and the disinfectant fully contact, and the shaking cleaning effect is more thorough.

[0024] The invention optimizes the process of in vitro rapid propagation of Cudrania chinensis by screening the components of the culture medium at each stage, thereby improving the conversion rate of the rapid propagation system. The method for establishing a tissue culture regeneration system of Cudrania chinensis is simple to operate, has a low pollution rate, a high survival rate, and a short culture cycle, and at the same time improves the reproduction efficiency of the rapid propagation system, which is helpful to produce a large number of high-quality seedlings in a short period of time, meet market demand, and promote the rapid development of Cudrania chinensis industrialization. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0026] Figure 1 This is the situation of inducing primary bud germination in Cudrania tricuspidata stem segments;

[0027] Figure 2 This is the induction of clustered shoot proliferation in Cudrania tricuspidata;

[0028] Figure 3 This is the rooting induction situation of Cudrania tricuspidata. DETAILED DESCRIPTION

[0029] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0030] It should be understood that the terms described in the present invention are only for describing a particular embodiment and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. The intermediate value in any stated value or stated range, and each smaller range between any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0031] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0032] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to those skilled in the art. The present invention description and examples are exemplary only.

[0033] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0034] Example 1

[0035] 1) Obtaining sterile materials

[0036] In April, cut the healthy and pest-free young branches of the mulberry tree of the year, cut the branches into stem segments of about 1.5 to 2.0 cm in length, ensure that there is a full bud on each stem segment, and the bud growth position is located at the upper 1 / 3 of the stem segment. Put the cut stem segments into a glass beaker and place them under running water for 1 hour. Soak them in 800 times carbendazim solution for 30 minutes, then soak them in 10wt% 84 disinfectant for 1 minute, rinse the stem segments with distilled water 5 times, and put them in a clean bench. Soak them in 75% volume concentration alcohol solution for 30 seconds, then immerse them in 0.1% mercuric chloride solution (add 3 drops of Tween 80 per liter) and oscillate them in a small ultrasonic cleaner (Jiemeng JP-008) for 9 minutes, rinse them with sterile water 5 times, and use sterile filter paper to absorb the moisture on the surface of the stem segments.

[0037] 2) Inducing bud germination from stem segments

[0038] The obtained stem segment explants were inoculated into a bud induction medium, the composition of which was as follows:

[0039] MS + 0.5 mg / L 6-BA + 0.01 mg / L IBA + 30 g / L sucrose + 7 g / L agar, pH adjusted to 5.8. Culture for 15 to 20 days at a light intensity of 2800 Lux, a culture room temperature of 25 ± 2 ° C, and a photoperiod of 16 h / d ( Figure 1 ).

[0040] 3) Cluster bud proliferation culture

[0041] Cut the tender shoots of the primary induction to obtain sterile seedlings and inoculate them into the cluster bud proliferation medium. The components of the medium are as follows:

[0042] MS + 2.0 mg / L 6-BA + 0.3 mg / L IBA + 25 g / L sucrose + 8 g / L agar, pH adjusted to 5.8. Culture for 30 to 45 days at a light intensity of 2800 Lux, a culture room temperature of 25 ± 2 ° C, and a photoperiod of 16 h / d ( Figure 2 ).

[0043] 4) Root induction of adventitious buds

[0044] The obtained buds were cut off and transferred to the rooting medium for rooting culture: well-induced buds were taken out under sterile conditions, buds with a stem length of 1.0-1.5 cm were cut off, and inoculated on the rooting medium for inducing adventitious roots. The rooting medium used 1 / 2MS as the basic medium, with 0.5 mg / L 6-BA, 0.5 mg / L IBA, 0.05 mg / L NAA and 2 mg / L mannitol added, 25 g / L sucrose and 8 g / L agar added to the medium, and the pH was adjusted to 5.8. Cultured for 20-30 days (with a light intensity of 2800 Lux, a culture room temperature of 25±2°C, and a light condition of 16 h / d) Figure 3 ).

[0045] In this example, 100 Cudrania tricuspidata in vitro regeneration tissues were constructed using young branches and stem segments with buds as explants. Among them, 3 explants failed to successfully construct in vitro regenerated Cudrania tricuspidata seedlings due to the formation of callus tissue during the construction process, which affected the rooting. The remaining 97 were all successfully constructed and all survived after seedling hardening and transplanting.

[0046] Test Example 1

[0047] On the basis of Example 1, the oscillation time of ultrasonic cleaning was adjusted according to Table 1, and the other operations were the same as Example 1, and 50 young branches and stem segments with buds of Cudrania chinensis were respectively subjected to in vitro regeneration culture, and the survival rate of the explants during the culture process was statistically analyzed, and the results are shown in Table 1. It can be seen from Table 1 that when the oscillation time of ultrasonic cleaning was 9 minutes, the contamination rate was the lowest and the survival rate of the explants was the highest.

[0048] Table 1 Survival rate of explants under different ultrasonic treatment times

[0049]

[0050] Note: Different letters represent significant differences at the p<0.05 level, the same below.

[0051] Test Example 2

[0052] The basal culture medium and the content of plant growth hormone in the bud induction medium, the cluster bud proliferation medium and the rooting medium were adjusted as shown in Tables 2 to 4. The remaining operations were the same as in Example 1. 50 young branches of Cudrania mulberry with bud stem segments were subjected to in vitro regeneration culture. The germination rate of the primary induction of the stem segments, the proliferation coefficient of the adventitious bud cluster proliferation culture and the rooting rate of the adventitious roots were statistically analyzed during the culture process. The results are shown in Tables 2 to 4.

[0053] Table 2 Germination of primary induction of stem segments

[0054]

[0055] Table 3 Adventitious bud cluster proliferation culture

[0056]

[0057]

[0058] Table 4 Adventitious root induction culture

[0059]

[0060] It can be seen from the data in Tables 2 to 4 that, considering the germination rate of primary induction of stem segments, the proliferation coefficient of adventitious bud cluster proliferation culture and the rooting rate of adventitious root induction, the most suitable basal culture medium in bud induction medium is MS, and the most suitable hormone content is 0.5 mg / L 6-BA + 0.01 mg / L IBA; the most suitable basal culture medium in cluster bud proliferation culture is MS, and the most suitable hormone content is 2.0 mg / L 6-BA + 0.3 mg / L IBA; the most suitable basal culture medium in rooting culture medium is 1 / 2MS, and the most suitable hormone content is 0.5 mg / L6-BA + 0.5 mg / L IBA + 0.05 mg / LNAA.

[0061] The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. A method for establishing an efficient regeneration system for Cudrania tricuspidata stem segments, characterized in that: The following steps are involved: Healthy young branches of Cudrania zedoaria tree of the current year were cut into stem segments, each of which had a full bud, and after preliminary disinfection, they were immersed in disinfectant and ultrasonically treated for disinfection before being used as explants; Inoculating the explants into a bud induction medium for bud induction culture to obtain young buds of sterile seedlings; Cutting the tender shoots of the sterile seedlings, inoculating them into a clustered shoot proliferation medium for proliferation culture, and obtaining the proliferated clustered shoots; cutting off the proliferated clustered buds, transferring them to a rooting medium for rooting culture, and obtaining in vitro regenerated seedlings of Cudrania tricuspidata; The components of the bud induction medium are MS+0.5mg / L 6-BA+0.01mg / L IBA+30g / L sucrose+7g / L agar; The components of the clustered bud proliferation medium are MS+2.0mg / L 6-BA+0.3mg / L IBA+25g / L sucrose+8g / L agar; The components of the rooting medium are 1 / 2MS+0.5mg / L 6-BA+0.5mg / L IBA+0.05mg / L NAA+25g / L sucrose+8g / L agar.

2. The establishment method according to claim 1, characterized in that: The length of the stem segment is 1.5 to 2.0 cm.

3. The establishment method according to claim 1, characterized in that: The ultrasonic treatment time is 9 minutes.

4. The establishment method according to claim 1, characterized in that: The preliminary disinfection includes flushing with tap water, soaking with carbendazim solution, soaking with 84 disinfectant and soaking with alcohol solution in sequence.

5. The establishment method according to claim 1, characterized in that: The disinfectant is a mercuric chloride solution, and the mercuric chloride solution contains Tween 80.

6. The establishment method according to claim 1, characterized in that: In the rooting culture stage, the clustered buds cut are buds with a stem length of 1.0 to 1.5 cm.

7. The establishment method according to claim 1, characterized in that: The pH values ​​of the bud induction medium, the clustered bud proliferation medium and the rooting medium are all 5.

8.

8. The establishment method according to claim 1, characterized in that: The culture conditions of the bud induction culture, the proliferation culture and the rooting culture are all 2800 Lux of light intensity, 25±2° C. of temperature and 16 h / d of photoperiod.

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